AOCS Methods for Biodiesel Feedstock Quality

Official Methods and Recommended Practices of the AOCS

AOCS Methods For Biodiesel Feedstock QualityGuidelinesCk 1-07 Recommended Practices for Assessing Feedstock to Ensure Biodiesel Quality

samplinGC 1-47 Sampling

CleanlinessCa 3a-46 Insoluble ImpuritiesCa 3d-02 Determination of Sediment in Crude Fats and Oils Centrifuge Method

impuritiesAcidity Cd 3d-63 Acid ValuePhosPhorus Ca 12-55 PhosphorusCa12a-02 Colorimetric Determination of Phosphorus Content in Fats and OilsCa 19-86 Phospholipids in Vegetable Oils Nephelonetric MethodCa 20-99 Analysis for Phosphorus in Oil by Inductively Coupled Plasma Optical Emission Spectroscopy sulfur Ca 17-01 Determination of Trace Elements in Oil by Inductively Coupled Plasma Optical Emission SpectroscopyWAter Ca 2e-84 Moisture Karl Fischer ReagentCa 2f-93 Determination of Moisture and Volatile Matter in Fats and Oils Modified Method

purityCa 6b-53 Unsaponifiable MatterCc 17-95 Soap in Oil Titrimetic MethodCd 20-91 Determination of Polar Compounds in Frying FatsCd 22-91 Determination of Polymerized Triglycerides by Gel-Permeation HPLC

Oxidative stabilityCd 12b-92 Oil Stability IndexCd 18-90 p-Anisidine ValueCd 20-91 Determination of Polar Compounds in Frying FatsCd 22-91 Determination of Polymerized Trigylcerides by Gel-Permeation HPLCCd 8b-90 Peroxide Value Acetic Acid Isooctane Method

Fatty aCid COmpOsitiOnCe 1-62 Fatty Acid Composition by Gas ChromatographyCe 2-66 Preparation of Fatty Acid Methyl EstersCd 1c-85 Calculated Iodine Value

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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

AOCS Recommended Practice Ck 1-07Approved 2007

Recommended Practices for AssessingFeedstock to Ensure Biodiesel Quality

DEFINITIONThis Recommended Practice lists those methods which may be used to assess the quality of oils andfats used in the production of biodiesel.SCOPEThis Recommended Practice is restricted to triglyceride feedstocks for biodiesel manufacture, includ-ing vegetable oils (soybean oil, rapeseed oil, palm oil, etc.), animal fats (tallow, lard, etc.) andtriglyceride greases (yellow grease, etc.). Critical attributes of the triglycerides are considered in lightof the requirements of the most common biodiesel process: the alkaline transesterification of thetriglyceride feedstock with methanol to produce fatty acid methyl esters. Only AOCS OfficialMethods or Recommended Practices are listed in Table 1; however, other procedures are reported inthe scientific literature.

Table 1Methods to assess feedstock quality.

Test Method

Sampling AOCS C 1-47Insoluble impurities AOCS Ca 3a-46Sediment by centrifugation AOCS Ca 3d-02Unsaponifiable matter AOCS Ca 6b-53Polar compounds in frying fats AOCS Cd 20-91Soap in oil AOCS Cc 17-95Polymerized triglycerides by gel-permeation HPLC

AOCS Cd 22-91Water by modified Karl Fischer method AOCS Ca 2e-84Modified moisture and volatiles AOCS Ca 2f-93 Acid value AOCS Cd 3d-63Sulfur, elements in oil by ICP-OES (use general guidance)

AOCS Ca 17-01Phospholipids in vegetable oils AOCS Ca 19-86Phosphorus in oil by ICP-OES AOCS Ca 20-99Phosphorus content, colorimetric method; AOCS Ca 12a-02Phosphorus AOCS Ca 12-55Fat stability, Oil Stability Index (OSI) AOCS Cd 12b-92Fat stability, peroxide value AOCS Cd 8b-90p-Anisidine value AOCS Cd 18-90Polymerized triglycerides by gel-permeation HPLC

AOCS Cd 22-91Polar compounds in frying fats AOCS Cd 20-91Fatty acid composition AOCS Ce 1-62 Methyl ester preparation AOCS Ce 2-66Calculated iodine value AOCS Cd lc-85

FACTORS TO LOOK FOR IN THE TRIGLYCERIDEFEEDSTOCK TO BE USED IN THE TRANSESTERIFICATION REACTIONRefined vegetable oils destined for food use are an expen-sive substrate for biofuel development. Although there isinterest in developing specialty oil crops, some producershave turned to less desirable or non-edible materials suchas animal fats and recycled vegetable oils. A key issue in

the production of biodiesel is the amount of intermediates,byproducts, unreacted substrates and a variety of contami-nants which may affect engine performance. Regional stan-dards provide some guidance on the methods of analysis tobe used, but do not address the analysis of the feedstock.Refined oil quality may be assessed using the OfficialMethods and Recommended Practices of the AOCS.Rendered animal fats and recycled greases are categorizedby grade. Each grade has detailed specifications which canbe determined by AOCS methods (for further details andexplanation see Notes and Table 2).

SAMPLING1. Fat products are frequently solids or semisolids at room

temperature and should be completely liquified andblended prior to testing. Temperature during meltingshould not exceed the melting point of the sample bymore than 10C.

2. Sample material in accordance with AOCS Method C1-47.

CLEANLINESS1. The feedstock should be free of trash or other foreign

material.2. Possible tests: Insoluble Impurities Ca 3a-46; Sediment

in Fats and Oils by Centrifugation Ca 3d-02

PURITY1. Composition shall be primarily triglyceride in nature.

Extensive levels of unsaponifiable material will lowerthe methyl ester content and possibly cause problems inbiodiesel processing. Regional mandates may requirethe methyl ester content in biodiesel to be a minimumof 96.5%.

2. Possible tests: Unsaponifiable Matter Ca 6b-53; PolarCompounds in Frying Fats Cd 20-91; Soap in Oil Cc17-95; Polymerized Triglycerides by Gel-PermeationHPLC Cd 22-91

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IMPURITIESWATER1. Excessive levels of moisture in the feedstock

will reduce catalyst effectiveness, will reducethe total conversion, and will yield undesirablebyproducts (soaps).

2. Possible tests: Modified Karl Fischer MethodCa 2e-84; Modified Moisture and Volatiles Ca2f-93

ACIDITY1. High acidity in the feedstock will reduce cata-

lyst effectiveness, will reduce total conversion,and will yield undesirable byproducts (soaps).If the acidity is high enough, alkaline transes-terification will not proceed.

2. Possible test: Acid Value of Fats and Oils Cd3d-63

SULFUR1. Limiting the sulfur content of the feedstock

will allow the biodiesel manufacturer to moreeasily meet the stringent specification on sul-fur (15 ppm S) in Ultra Low Sulfur Dieselfuels without additional expensive after-treat-ment.

2. Possible test: Trace Elements in Oil by ICP-OES Ca 17-01

PHOSPHORUS1. Phosphorus is an indication of the presence of

phospholipids in feedstocks from plant origins.During vegetable oil refining, phospholipidsare normally removed. If a significant amountof phospholipid remains, it will cause prob-lems in the biodiesel transesterification andpurification steps. If the phosphorus is notremoved from the FAME product, it will notmeet the phosphorus limit (10 ppm P) allowedin biodiesel. It is easier to start with a feed-stock already low in phosphorus than to try toremove it in after-treatment.

2. Possible tests: Phospholipids in Vegetable OilsCa 19-86; Phosphorus in Oil by ICP-OES Ca20-99; Phosphorus Content, ColorimetricMethod Ca 12a-02; Phosphorus Ca 12-55

OXIDATIVE STABILITY1. The stability of the triglyceride feedstock will have

great bearing on the oxidative stability of the biodieselprepared.

2. Possible tests: Fat Stability, Oil Stability Index (OSI)Cd 12b-92; Fat Stability, Peroxide Value Cd 8b-90; p-Anisidine Value Cd 18-90; Polymerized Triglyceridesby Gel-Permeation HPLC Cd 22-91; Polar Compoundsin Frying Fats Cd 20-91

FATTY ACID COMPOSITION1. Analysis of the fatty acid composition is useful addi-

tional information which may indicate the source of thefat or oil being used as feedstock. The iodine value ofthe feedstock may be determined by calculation.

2. Possible tests: Fatty Acid Composition Ce 1-62;Preparation of Methyl Esters Ce 2-66; CalculatedIodine Value Cd 1c-85.

NOTES

Table 2. AOCS Methods for Tallow and Grease QualityParameters Recognized by the National RenderersAssociation.

For an explanation of the tests, refer to 115, below.1. Boehmer number Cb 5-40, surplus*2. a. Color (FAC) standard Cc 13a-43*

b. R & B Color Cc 8d-55*3. Fatty acid profile Ce 1-624. Free fatty acids (FFA as OA) Ca 5a-405. Iodine value (IV) Cd 1d-926. Lead content Ca 18c-917. Moisture, impurities, unsaponifiables (MIU)

a. Moisture Ca 2c-25 or Ca 2b-38b. Unsaponifiable matter Ca 6a-40c. Insoluble impurities Ca 3a-46

8. Peroxide value (PV) Cd 8b-909. Pesticide residue Use EPA/FDA methods**10. pH 11. Polyethylene (PE) Ca 16-7512. Rate of filtration 13. Saponification value (SV) Cd 3-2514. Titer Cc 12-5915. Total fatty acids (TFA)

* Methods in normal, and bold text are current or recom-mended for use in the Tallow and Grease Series of theAOCS Laboratory Proficiency Program (LPP), respec-tively.

** Only required for food grade or feed grade materials

Rendered fats are defined by the following tests: 1. Boehmer number refers to a test to find out whether tal-

low is mixed in with lard. If the number is less than 73,contamination has occurred.

2. Color is quantified by comparing a sample of filteredliquid fat to the Fat Analysis Committee (FAC) stan-dard and assigning it a number from 1 (lightest) to 45(darkest). R & B Color means refined and bleached. Forexample, the specification for extra fancy tallow is usu-ally one Red, but is sometimes specified at 0.5 Red.

3. Fatty acid profile is the relative amounts of the 16 pos-sible fatty acids as determined by gas chromatography.

4. Free fatty acids (FFA), the amount of fatty acids splitfrom the triglyceride or fat molecule and dissolved inthe fat. FFA are a measure of the hydrolysis that hastaken place within the fat molecule. Time, temperature,and the presence of moisture, bacteria, and enzymesinfluence the hydrolysis of fat into free fatty acids andglycerol.

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5. Iodine value (IV) is a measure of the chemical unsatu-ration of the fat. It is expressed as the number of gramsof iodine absorbed by 100 g of fat sample.

6. Lead content is determined by automatic analyzer. Thetolerance level for lead is 7 ppm (mg/kg), above whichit is considered toxic.

7. Moisture, impurities, unsaponifiables (MIU) should notexceed 1 to 2%, depending on how the fat is to be used.

a. Moisture in fat arises from slight emulsifica-tion during processing and is determined bydistillation with toluene or by heating; itshould be 0.5 to 1%.

b. Unsaponifiables are any material that will notsaponify (form soap) when mixed with analkali. They are soluble in ordinary fat solventsand include sterols, pigments, and hydrocar-bons. These particles are inherent in all fats,both animal and vegetable, and may arise fromcontamination with petroleum products.

8. Peroxide value (PV) is an indication of stability andrancidity.

9. Pesticide residue must not exceed defined levels forcertain chemicals that are toxic to animals; 0.5 ppm forDDT, DDD, and DDE; 0.3 ppm for dieldrin; 2.0 ppmfor PCB. The analysis is done by gas chromatography.(Note, the allowable limits frequently change; refer tothe Federal Register for current limits.) These tests arenot relevant to biodiesel production.

10. pH is determined on a scale of 0 to 14: 7 is neutral,below 7 is acid, above 7 is alkaline.

11. Polyethylene (PE) is a foreign material in tallow, whichfinds its way into the rendering plant as meat wrappersmixed in with raw material.

12. Rate of filtration is an analytical method in which agiven volume of liquid fat sample will filter in a speci-fied time under standard conditions. Filtration is slowedby the presence of fine particles and glue substances;the rate of filtration indicates whether a batch of fat willgive processing difficulties.

13. Saponification value (SV) is an estimate of the meanmolecular weight of the constituent fatty acids in a fatsample. It is defined as the number of milligrams ofpotassium hydroxide required to saponify 1 g of fat.The higher the SV, the lower the mean chain length ofthe triglycerides.

14. Titer is the solidification point of the fatty acids, animportant characteristic in fats used to produce soap orfatty acids. Trade practice is to designate animal fatswith titers of 40 C and up as tallow and those below 40C as grease.

15. Total fatty acids (TFA) both the free fatty acids andthose combined with glycerol (intact glycerides) shouldexceed 90% by weight. Fat is composed of approxi-mately 90% fatty acids and 10% glycerol. The caloriecontent for glycerol is about 4.32 per gram comparedwith 9.4 for fatty acids. Since fatty acids contain morethan twice the energy of glycerol and some other fattysubstances, the TFA content of fat indicates energycontent. This may be determined from the fatty acidcomposition when using an internal standard.

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AOCS Official Method C 1-47Revised 1999, 2000

SamplingSCOPEAp p l i c able to the sampling of animal and vege t able fats, and crude and re fined vege t able and marine oils.

APPARATUS1 . C o re sampler for oils and liquid fats (official trier of the

N ational Cottonseed Products Association) (Fi g. 1)ametal tube of 5.0-cm (2-in.) diameter throughout. Th elength must be sufficient to take a cross section thro u g hthe entire depth of oil, about 3 meters (10 ft) for tankc a rs. One end of the trier is fitted with a tight va l vewh i ch allows an unre s t ricted opening 5.0 cm (2 in.) in

diameter when fully opened and is free from leakswhen cl o s e d. The va l ve is opened and closed by meansof a rod from the top of the tri e r. The trier is soc o n s t ructed as to take a sample within 6.4 mm (0.25 in.)(or less) of the bottom of the tank. The sampler is suit-able for tank cars .

2 . Bomb or zone sampler for oils and liquid fats (Fi g. 2)a tightly closed cy l i n d rical compartment so constru c t e dt h at a sample can be taken from any specified section ofthe tank. The sampler must permit taking a sample fro mwithin 13 mm (0.5 in.) (or less) of the bottom of thetank. The va l ve or va l ves must be tight-fitting so thesampler can be withdrawn without loss or tra n s fer ofcontents. The va l ves should be re a d i ly opened by handand manipulated either automat i c a l ly or by an at t a ch e dc o rd. The device must be re a d i ly cl e a n abl e.

3 . Trier for solid fatsa half-round metal tube 1325 mm(0.51 in.) in diameter. The length may be 61213 cm(27 ft), depending upon the size of pack age to bes a m p l e d. One end is tap e red to a point, the taper to benot more than 2.5 cm (1 in.) long. The other end isat t a ched to a D- or T- s h aped handle. Copper, brass, orb ro n ze must not be used in the construction of this tube.Figure 1. Core sampler for oils and liquid fats (see Notes, 4).

Figure 2. Liquid zone sampler (see Notes, 5).

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NoteA trier for tank cars when the contents aresolid may be similar but about 5.0 cm (2 in.) in diame-ter and 22.5 meters (78 ft) long.

4 . Oil thiefa glass tube 9.513 mm (3812 in.) i.d. Th elength may be convenient for the size of pack ages to bes a m p l e d. One end is constricted to about 6.5 mm bymeans of a short taper no longer than 2.5 cm. The otherend is constricted suffi c i e n t ly so it can be used as afi n ger va l ve.

NoteIf the material to be sampled is semisolid,the constriction at the lower end must be enlarged suf-ficiently to permit the material to flow into the tube.

5 . Sample containers as necessary (see Notes, 1).

SIZE AND NUMBER OF SAMPLES1 . The ge n e ral pro c e d u re is to draw a number of port i o n s

f rom the bulk quantity or a number of portions from allor seve ral pack ages, composite these, mix thoro u g h lyand distri bute rep re s e n t at ive portions into suitably size da i rtight containers for the lab o rat o ry sample. Th enumber of lab o rat o ry samples to prep a re depends onthe circumstances. In all cases involving commerc i a lt ransactions, a minimum of three (pre fe rably four) isre q u i re d. This permits one for the bu ye r, one for theseller and one or two in re s e rve for possible arbitrat i o n( re fe ree analy s i s ) .

2 . A gross sample in the pro p o rtion of not less than 50 lb( about 23 kg) per 60,000 lb cargo is re q u i red of all tankc a rs, and a minimum of 50 lb (about 23 kg) is re q u i re dfor all other bulk oil quantities, such as in ships or shoret a n k s .

3 . A gross sample in the pro p o rtion of not less than 20 lb(9 kg) for each 100 barrels or equivalent quantity isre q u i red when drums, tierces, barrels and other pack-ages are sampled.

4 . When sampling drums, tierces and all other pack age s ,e a ch pack age is to be sampled unless the concern e dp a rties agree to the fo l l owing sch e d u l e :

Number of packages Number of packagesin shipment to be sampled*

110 131025 242550 365075 6875100 810

*In no case should less than 10% of the packages be sampled.

5 . In the case of all bulk oil shipments, whether in dru m s ,tank cars, ships or shore tanks, the minimum size fo re a ch lab o rat o ry sample is about 4 L (1 ga l ) .

6 . In the case of tallows, greases and other inedible fat s ,the minimum size for each lab o rat o ry sample is ab o u tl.3 kg (3 lb).

7 . In the case of edible fats and oils, the minimum size fo re a ch lab o rat o ry sample is about l kg (2 lb) for fats and 1L (1 qt) for oils. If re fining or bl e a ching tests arere q u i re d, the minimum quantity is about 4 L (1 ga l ) .

GENERAL PRINCIPLES1 . It is impossible to write directions for sampling fats and

oils that will encompass all conditions and circ u m-stances that may confront the individual ch a rged withthe responsibility of taking the sample. Th e re are manyinstances in wh i ch the ex p e rience and judgment of thati n d ividual must prevail. Th e re are, howeve r, cert a i nge n e ral rules wh i ch must always gove rn if the sample isto be rep re s e n t at ive.

2 . The best sample of bulk oil quantities can be taken ifthe product to be sampled is completely liquid and thor-o u g h ly mixe d. In such cases, a core sample or even asample dipped from the tank while undergoing vigo r-ous agi t ation will be rep re s e n t at ive.

3 . Settled mat e rial, in wh i ch the water and solid impuri t i e sa re like ly to be concentrated at the bottom, is difficult tosample and reconstitute in pro p o rtional quantities. Th econtour of the tank must be taken into account. If thebottom of the tank is smaller than the middle or top, orvice ve rs a , a core sample is not adequat e. To ove rc o m ethis, the number of portions from each section [e. g. ,e a ch 30-cm (1-ft) level] should be inve rs e ly pro p o rt i o n a lto the cubical capacity of each section. For ex a m p l e, ifthe bottom 1-ft section is one-fo u rth of the middle 1-ftsection, then one 1-ft sectional sample should be draw nf rom the bottom level and four 1-ft sectional samplesf rom the middle section. These portions are thencomposited into one sample and mixe d.

4 . All samples must be completely labeled for identifi c at i o n .

PROCEDURE1 . C o n t i nu o u s - fl ow method (Fi g. 3) for sampling tank and

tank car during loading or unloading(a) If the conditions are suitable, this is a satisfactory

method of sampling. This method is ap p l i c abl eo n ly if the product is completely liquid and fre efl owing and does not contain any mat e rial thatmay plug the bleeder line.

(b) The 38-in. bleeder line is 3/8-in. standard pipe witha slight dow n wa rd slope, located in a ve rt i c a lsection of the pumping line through wh i ch thep roduct is continu o u s ly fl owing upwa rd to the indi-vidual tank or tank car being sampled. The sampleline should be located as far away from elbows ortees as possibl e, should penetrate to the center ofthe pumping line, should be cut beveled at the endlooking dow n wa rd, and should disch a rge into asample tank or drum as illustrated in Fi g u re 3. Th esample line should not have a petcock .

(c) The metal sample tank or drum is of ap p rox i-m at e ly 50-gal (185-L) cap a c i t y. Just ab ove thebottom of the drum a 38-in. draw-off line equippedwith petcock is installed and is used for obtainingthe required sample(s) from the gross sample. Tofacilitate complete draining and easy cleaning, thebottom of the drum should be replaced by as e c u re ly welded inve rted-cone bottom having anap ex angle of ap p rox i m at e ly 120, the other twoangles with the hori zontal being about 30 each .To prevent loss of solvent by evaporation, a suit-

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able metal cove r, with slots or holes to perm i tinsertion of sampling pipe and mixer shaft, shouldbe placed over the sample tank during thesampling and mixing operation.

(d) P rior to the start of the pumping peri o d, thesampling equipment should be examined and thedraw-off lines closed. During the pumping periodit should be made certain that a continuous flow ofoil is being obtained. When the filling of the tankor tank car has been completed, the mech a n i c a lm i xer is started and the gross sample is mixe dthoroughly in order to obtain uniform distribution

of moisture, oil and impurities. After thoro u g hm i x i n g, with the agi t ator still ru n n i n g, the draw -off line is opened and three 1-gal (3.7-L) samplesa re withdrawn into new and dry 1-gal (3.7-L)c o n t a i n e rs to a level about 5 cm (2 in.) from thetop. The sample containers are immediately closedand properly labeled.

(e) S u rplus oil remaining in the sample tank isre t u rned to oil storage, or to the tank car if theo fficial weight was obtained prior to sampling,through the 3-in. line connected to the apex of theconical bottom. After dra i n i n g, the tank is thor-

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C 1-47 Sampling

Figure 3. Schematic of sampling device.

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o u g h ly cleaned by fl u s h i n g, washing and dry i n g,or other suitable means, depending upon condi-tions, and the cleanings withdrawn through theclean-out line. The cleaning pro c e d u re should bes u ch that there will be no contamination of thenext sample drawn.

N o t e Wh e re multiple loading or unloadingfacilities are in use, a separate sample tank shouldbe available for each unit.

(f) In locations where the liquid pressure in the 3-in.line is too high to retain all of the sample in the50-gal (185-L) drum, an intermittent sample maybe taken. For this purpose, install a 3/8-in. electricsolenoid valve as shown in Figure 3. It may be aslide gate va l ve wh i ch opens fully and does nothave any area to retain solids. The valve is oper-ated by a timer. With a 90 psig and normal pump-ing rate, the valve is open 30 sec and closed 25 secto secure 50 gal (185 L) per 60,000 lb oil. Th evalve should be operated to open and close at leastone time every minute.

2 . G rab method for sampling tanks or tank cars duri n gloading or unloading(a) Use a dipper, or any other convenient container,

and withdraw about 1 lb (454 g) from the disch a rgeend of the pipe at regular intervals as the product ise n t e ring the tank.

(b) Collect, mix and distri bute as directed in Pro -cedure, 1, (d).

3 . Loaded tanks or tank cars, liquid contents (official methodof the National Cottonseed Products Associat i o n ) (a) L ower the official oil trier ve rt i c a l ly through the oil

at a unifo rm rate with the bottom va l ve completelyopen so that 1015 sec will be re q u i red to re a ch thebottom of the car (see Notes, 2). Close the bottomva l ve and withdraw the tube.

(b) Ta ke seve ral portions in this manner and thenproceed as directed in Procedure, 1, (d).

4 . Loaded tanks or tank cars, solid contents(a) Solid mat e rial cannot be corre c t ly sampled in

tanks or tank cars. If possible, the material shouldbe liquified (see Notes, 3) and then sampled asdirected in Procedure, 3.

(b) When necessary to sample solid mat e rial (see Notes,3), use the designated trier and withdraw seve ra lp o rtions from the car taken ve rt i c a l ly and obl i q u e lyt owa rd the ends of the car. The trier should passt h rough the stock until it touches the sides of the carso a complete core will be taken. Soften (but do notmelt) and mix all portions thoro u g h ly befo re distri b-uting into lab o rat o ry sample containers .

5 . Ship and shore tanks, liquid contents, using a bomb orzone sampler(a) Tanks constructed so that the sampler cannot be

l owe red ve rt i c a l ly to the lowest part of the tankcannot be corre c t ly sampled for moisture andsettlings with any sampler. In order to sample suchm at e rial pro p e rly, the contents should be pumped toanother tank into wh i ch the sampler can be lowe re dto the lowest part of the tank. The sampling may

then be done by the petcock method during pump-ing or with a suitable sampler after the tra n s fe r.

(b) If the tanks are suitably constru c t e d, lower thesampler to the lowest point in the tank. As soon asthe sampler is completely full, withdraw and emptyinto a suitable container. If any fo reign mat e ri a l( wat e r, dirt, etc.) is encountere d, take samples atc o n s e c u t ive 2.5-cm (1-in.) levels until there is nof u rther evidence of these impurities. Above this leve lw i t h d raw samples at consecutive 30-cm (1-ft) leve l s ,if the sampler is a 30-cm (1-ft) sampler, until the topl evel of the oil has been re a ch e d. The samples arecomposited in the pro p o rtion that each rep resents tothe total depth of oil in the tank. For ex a m p l e, if 12p o rtions are taken at 2.5-cm (1-in.) levels and thetotal depth is 6 m (20 ft), combine in the ratio of 1 ofthe portions taken at the 2.5-cm (1-in.) levels to 19 ofthe portions taken at higher leve l s .

(c) Combine 11 portions and mix them thoro u g h lytogether before distribution into laboratory samplecontainers.

6 . Ship and shore tanks, liquid contents, using a cores a m p l e r (a) Use a sampler as described in Apparatus, 1, except

of sufficient length to take a cross section throughthe entire depth of oil. This method is impracticalif the tanks to be sampled are so deep as to makethe sampler unwieldy. In such cases, the sectionalsampler must be used.

(b) Proceed as directed in Procedure, 3.7 . B a rrels, tierces, casks and drums; liquid or semisolid

c o n t e n t s (a) Roll the container to mix the contents, and insert

the oil thief slow ly (see Notes, 2 and 3) thro u g hthe bung or, preferably, through an end opening orhole drilled at one end. If possibl e, the sampleshould be drawn from end to end.

(b) As soon as the thief is fully insert e d, close theupper constriction with a fi n ger and tra n s fer thesample into a suitable container. Ta ke seve ra lportions in this manner from this and other pack-ages as directed in Size and Number of Samples,4. Mix thoroughly before distribution into labora-tory sample containers.

8 . B a rrels, tierces, casks and drums; solid contents(a) R e m ove the bung or end opening, or drill a hole

through an end or side with an auger large enoughin diameter to accommodate the trier (see Notes,3). If possibl e, the sample should be drawn fro mend to end.

(b) I n s e rt the trier through the opening, push it thro u g hto the opposite end or side, turn it in a completec i rcle and withdraw with the sample. Collects eve ral such portions from this and other pack age sas directed in Size and Number of Samples, 4.Soften (but do not melt) and mix thoro u g h ly befo red i s t ri bution into lab o rat o ry sample containers .

9 . B a rrels, tierces, casks and bags; ve ry hard mat e ri a l s (a) If the mat e rial is in the fo rm of fl a kes or small

pieces, take grab samples of uniform and propor-

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tional size from packages as directed in Size andNumber of Samples, 4. If the mat e rial is in thefo rm of large pieces, these should be bro ken upb e fo re taking the grab sample. Mix thoro u g h ly,q u a rter and distri bute into lab o rat o ry samplecontainers (see Notes, 3).

l 0 . Pa ck ages of odd sizes and mixed lots(a) The procedure for taking samples and the number

of packages to be sampled is exactly as describedin Procedure, 7, 8, and 9.

NOTESCautionS u l f u ric acid is a strong acid and will cause seve re bu rn s .P ro t e c t ive clothing should be wo rn when wo rking with thisa c i d. It is a dehy d rating agent and should not be stored in thevicinity of organic mat e rials. Use gre at caution in mixingwith water due to heat evolution that can cause ex p l o s ives p l at t e ri n g. Always add the acid to wat e r, never the reve rs e.

NUMBERED NOTES1 . If the stability or ke eping quality of the product is

i nvo l ve d, all equipment and containers must be scru p u-l o u s ly clean. The containers may be new (unused) tincans or glass jars. Metal, such as copper, bro n ze andb rass, must under no circumstances be allowed to comein contact with the sample. Glass containers are cl e a n e dwith an ap p ro p ri ate cleaning solution, thoro u g h lyrinsed with distilled water to re m ove all traces of cl e a n-ing solution, and dried by heat. Glass jars with ru bb e rga s kets are sat i s fa c t o ry, but all parts must be cleaned asd e s c ribed ab ove. Ja rs with plastic or enameled tops, orc ove rs containing paper liners, are not re c o m m e n d e d.

Prepare the cleaning solution by placing 510 g ofpotassium dichromate (K2Cr2O7) in a 1-L Erlenmeyerflask with about 50 mL of water. Warm the flask in a

hot water bath to dissolve as much dichromate as pos-s i bl e. Slow ly and care f u l ly add concentrated H2S O4(see Notes, Caution) until the volume is about 200 mL.Allow the hot solution to stand for about 5 min; thendilute to 1 L with concentrated H2SO4.

Remove sample from the package with a stainlesssteel trier [butter type, 4692 cm long (1836 in.)] thathas been previously well cleaned with soap and water,t h o ro u g h ly rinsed with distilled water and completelydried by heat or with a new paper towel. Samples arecollected so none of the shortening will be taken lessthan 5 cm (2 in.) from the well of the container or fromthe surface of the sample.

Samples are packed and transported to the destina-tion laboratory in such a way that they will be protect-ed from the light and arrive in a solid stat e. Samplest h at have been melted or part i a l ly melted at any timeare not satisfactory.

2 . As the trier is lowe red into the oil, the rate must be slowenough so that the level of oil inside and outside of thet rier remains the same. Otherwise, an unduly large port i o nwill be drawn from the bottom, wh i ch is like ly to containa considerable concentration of moisture and settlings.

3 . If the physical stru c t u re of the product is invo l ve d, such asthe consistency of shortening or the graininess of cert a i ntypes of animal fats and hy d roge n ated vege t able fats, them at e rial must not be softened, melted or sampled with anyof the devices mentioned herein. In such cases, the cove rof the container must be re m oved and a large section (ifs h o rtening) cut out with a large spatula without disturbingthe surfa c e. If the product is gra i ny, use a large dipper tore m ove the sample with as little disturbance as possibl e.

4 . The add ress for Refi n e ry Supply Co. is 6901 E. 12thSt., Tulsa, OK 74112, USA. Phone (918) 836-4681.

5 . The add ress for Zone Devices is 3449 Ocean Vi ew Blvd. ,G l e n d a l e, CA 91208 USA. Phone (818) 249-5887.

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C 1-47 Sampling

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AOCS Official Method Ca 3a-46Replacing Ca 3-25 and Ca 3-46 Reapproved 1997

Insoluble ImpuritiesDEFINITIONThis method determines dirt, meal and other foreign substances insoluble in kerosene and petroleumether.SCOPEApplicable to all normal fats and oils.

APPARATUS1 . G o o ch cru c i bl e p rep a red with a glass-fiber filter with-

out organic filler (Reeve Angel 934 AH or Wh at m a nGF/C). Wash the filter with wat e r, alcohol and ether.D ry to constant weight at 101 1C. Cool in a desicca-tor to room temperat u re and we i g h .

2 . Filter flask of convenient size and Gooch cru c i bl ea d ap t e r.

REAGENTS (see Notes, Caution)1 . Pe t roleum etherAOCS Specifi c ation H 2-41.2 . Ke ro s e n e re fined petroleum distillate with a fl a s h

point not below 23C (75F), as determined by theA m e rican Society for Testing and Mat e rials StandardMethod D56, using the tag closed tester. The ke ro s e n eshould be fi l t e red through a Gooch cru c i bl e, prep a re das in Ap p a ratus, 1, befo re using.

PREPARATION OF SAMPLE1 . Samples must be mixed thoro u g h ly. If necessary, soften

with gentle heat (do not melt) and mix thoro u g h ly withan efficient mixe r.

PROCEDURE1 . Use the residue from the moisture and vo l atile mat t e r

d e t e rm i n ation (AOCS Official Methods Ca 2b-38 or Ca2d-25), or a sample prep a red in the same manner (seeNotes, 1).

2 . A dd 50 mL of ke rosene to the residue and heat in awater bath to dissolve the fat .

3 . Filter through the prep a red Gooch cru c i ble with the aidof a vacuum. Wash with five 10-mL portions of hotke ro s e n e, allowing each portion to drain befo re add i n gthe nex t .

4 . Wash thoro u g h ly with petroleum ether to re m ove all ofthe ke ro s e n e. Dry the cru c i ble and contents to aconstant weight at 101 1C, cool to room temperat u rein a desiccator and we i g h .

CALCULATIONS1 . I n s o l u ble impurities, % =

gain in mass of crucible 1 0 0

mass of sample taken for moisture

NOTESCautionPe t roleum ether and ke rosene are fl a m m able solvents. Afume hood should be used at all times when working withthese solvents.

NUMBERED NOTES1 . Samples (e. g., certain feed stocks) with a higher than

usual perc e n t age of insoluble impurities may be diffi-cult, if not impossibl e, to fi l t e r. A study among fivel ab o rat o ries indicated that, for such samples, a 2-gsubsample (rather than a 5-g subsample as specified inAOCS Official Methods Ca 2b-38 and Ca 2d-25) can beused without gre at ly affecting the precision of them e t h o d.

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AOCS Official Method Ca 3d-02Approved 2002

Determination of Sediment in Crude Fats and OilsCentrifuge Method

DEFINITIONA homogenized test sample is subjected to centrifuging as specified. The amount of separated mater-ial, called sediment (part of the insoluble matter in a crude fat or oil which can be centrifugally sepa-rated and is the total amount of the unclear layer of components collected at the bottom of the mea-suring tube after centrifuging) is volumetrically measured in a calibrated centrifuge tube.SCOPEThis method determines sediment that can be separated from crude fats and oils by centrifugal force.The method is applicable to crude oils and to oils with a sediment content of 0.03 mL per 100 g to 15mL per 100 g, obtained by means of extraction and/or crushing. The method is not applicable to fatswhich are not liquid at a temperature of 20C. This method is identical to International Organizationof Standardization (ISO) 15301.

APPARATUSUsual laboratory apparatus and, in particular, the following:

1. Centrifuge tubes, 100 mL capacity , pear- or cone-shaped, made from thoroughly annealed glass and fit-ted with a stopper (see Figures 1 and 2) (see Notes, 2).

2. Buckets, for centrifuge tubes (Apparatus,1), resistant tofats and oils.

3. Centrifuge, suitable for the centrifuge tubes(Apparatus, 1) placed in the buckets (Apparatus, 2),capable of controlling the rotational frequency so as togive a radial acceleration at the narrow part of the

Figure 1. Pear-shaped sediment tube.

Figure 2. Cone-shaped sediment tube.

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tubes of 700 to 800 times the acceleration of free fall.See Notes, 3 for the calculation of the rotational fre-quency of the centrifuge. In rooms which are not air-conditioned, use a centrifuge capable of maintaining itstemperature between 20C and 25C.

4. Balance, capable of weighing to the nearest 0.01 g.

SAMPLING1. It is important that the laboratory receive a sample

which is truly representative and has not been damagedor changed during transport or storage.

2. A recommended sampling method is given in AOCSOfficial Method C 1-47 or ISO 5555 (1).

3. Store samples in glass or polyethylene terephthalate(PET) bottles.

TEST SAMPLE PREPARATION1. Prepare the test sample in accordance with ISO 661. 2. Bring the test sample, if necessary, to a temperature of

between 20C and 25C. 3. Redisperse any sediment in the oil from the bottom of

the sample bottle to ensure a sufficiently homogeneousand representative sample. Immediately proceed inaccordance with Procedure.

PROCEDURE1. Weigh two centrifuge tubes (Apparatus, 1) to the near-

est 0.1 g. 2. Transfer the prepared test sample (see Test sample

preparation, 13) to each of the centrifuge tubes. 3. Weigh the tubes and place them in the buckets

(Apparatus, 2) of the centrifuge (Apparatus, 3). Adjustspeed to give a radial acceleration at the narrow part oftubes of 700800 g.

4. Centrifuge for 1 h 5 s.5. Read sediment volumes up to and including 1.5 mL to

the nearest 0.03 mL. Read sediment volumes greaterthan 1.5 mL to the nearest 0.5 mL.

6. When using a cone-shaped tube, it may be more diffi-cult to read the volume of the unclear layer; read thesediment volumes as accurately as possible.

7. If in a pear-shaped tube the separation is not complete(clear layer in the neck of the narrow tube section ofthe tube), the sediment reading should be corrected forthis volume.

8. Record the g force (rpm) used or the swing diameterand the rpm of the centrifuge (Notes, 3).

9. Record the temperature before and after centrifuging.

EXPRESSION OF RESULTSCalculate the sediment content of the test sample using theequation:

V 100w =

(m1 m2)

Wherew = the numerical value of the sediment content of the

test sample, in milliliters per 100gV = the numerical value of the sediment volume, in

millilitersm1 = the numerical value of the mass of the centrifuge

tube with the test portion, in gramsm2 = the numerical value of the mass of the centrifuge

tube, in grams

Calculate the mean of the results for the two tubes andreport the results to the nearest 1 mL per 100 g.

PRECISION1. Details of interlaboratory tests on the precision of the

method are given in Tables 1 and 2. The values derivedfrom these tests may not be applicable to concentrationranges and matrices other than those given. The preci-sion of the method was established by two interlabora-tory tests organized by the Netherlands Oils, Fats andOilseeds Trade Association (NOFOTA) in cooperationwith the Federation of Oils, Seeds and FatsAssociations (FOSFA International) in 1996 and1997/1998 and carried out in accordance withInternational Organization of Standardization 5725-2(3). In the first test 12 laboratories participated. Six(spiked) samples of crude sunflower seed oil wereinvestigated. In the second test 9 laboratories partici-pated. Four (spiked) samples of crude sunflower seedoil were investigated. See Tables 1 and 2 for a summa-ry of the statistical results of the tests.

Comparison with the 96-h methodIn the two interlaboratory tests, the centrifuge method wascompared with the method which leaves the test sample tostand at a controlled temperature for 96 h. Applying regres-sion analysis to the results of both interlaboratory tests for

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Ca 3d-02 Determination of Sediment in Crude Fats and Oils

Table 1Statistical results of the interlaboratory test organized in 1996.

Parameter Sample

Number of laboratories retained after eliminating outliers 12 12 12 11 10 11Mean sediment content, mL per 100 g 0.75 1.36 0.54 1.62 2.07 2.61Repeatability standard deviation (sr), mL per 100 g 0.04 0.06 0.04 0.06 0.03 0.07Repeatability coefficient of variation, % 4.76 4.29 6.85 3.60 1.59 2.78Repeatability limit (r) [r = 2.8 sr], mL per 100 g 0.10 0.16 0.10 0.16 0.09 0.20Reproducibility standard deviation (sR), mL per 100 g 0.26 0.22 0.26 0.28 0.26 0.35Reproducibility coefficient of variation, % 34.6 16.0 47.9 17.2 12.6 13.5Reproducibility limit (R) [R = 2.8 sR], mL per 100 g 0.72 0.61 0.72 0.78 0.73 0.99

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the relation between the two methods, the following equa-tion was established (with a correlation coefficient of 0.96):

w2 = 2.5w1 0.5

w1 = the numerical value of the sediment contentobtained with the 96-h method, in milliliters per100 mL

w2 = the numerical value of the sediment contentobtained with the centrifuge method, in millilitersper 100 mL

This indicates that the centrifuge method may be consid-ered as a good alternative to the 96-h method, where arapid control method is required for levels of sedimentexceeding 0.5 mL per 100 mL.

2. RepeatabilityThe absolute difference between two independent singletest results, obtained using the same method on identicaltest material in the same laboratory by the same operatorusing the same equipment within a short interval oftime, will in not more than 5% of cases exceed therepeatability limit given in or derived from Table 3.

3. ReproducibilityThe absolute difference between two single test results,obtained using the same method on identical test mate-rial in different laboratories by different operatorsusing different equipment, will in not more than 5% ofcases exceed the R reproducibility limit given in orderived from Table 3.

NOTES1. The sediment contains, for example, phospholipids,

impurities, dirt, etc. dispersed in a water-containingphase, and can be quantified according to this method.Any white crystalline components deposited on top ofand within the dark layer of insoluble materials areregarded as part of the sediment.

2. Reading the volume of the unclear layer may be moredifficult in a cone-shaped tube than in a pear-shapedtube.

3. The relationship between centrifugation parameters isgiven by the equation:

arn = 1,337dWhere

n = the numerical value of the rotational frequency, inmin1;

ar = the relative radial acceleration (for example ar =700 or ar = 800);

d = the numerical value of the diameter of swing,measured between the tips of opposite tubes whentubes are in a rotating position, in millimeters.

REFERENCES1. ISO 5555:1991, Animal and vegetable fats and oils

Sampling. 2. ISO 5725-1:1994, Accuracy (trueness and precision) of

measurement methods and results Part 1: Generalprinciples and definitions.

3. ISO 5725-2:1994, Accuracy (trueness and precision) ofmeasurement methods and results Part 2: Basicmethod for the determination of repeatability andreproducibility of a standard measurement method.

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Ca 3d-02 Determination of Sediment in Crude Fats and Oils

Table 2Statistical results of the interlaboratory test organized in 1997/1998.

Parameter Sample

Number of laboratories retained after eliminating outliers 7 8 8 7Mean sediment content, mL per 100 g 0.07 1.28 1.20 2.39Repeatability standard deviation (sr), mL per 100 g 0.00 0.04 0.04 0.00Repeatability coefficient of variation, % 0.00 2.77 2.95 0.00Repeatability limit (r) [r = 2.8 sr], mL per 100 g 0.00 0.10 0.10 0.00Reproducibility standard deviation (sR), mL per 100 g 0.07 0.12 0.08 0.35Reproducibility coefficient of variation, % 97.8 9.1 7.0 14.8Reproducibility limit (R) [R = 2.8 sR], mL per 100 g 0.20 0.33 0.24 0.99

Table 3Repeatability limit (r) and reproducibility limit (R ).

Sediment content r R

mL per 100 g mL per 100 g mL per 100 g< 1 0.1 0.71 to 3 0.2 1.0

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AOCS Official Method Cd 3d-63Revised 2003

Acid ValueDEFINITIONThe acid value is the number of milligrams of potassium hydroxide necessary to neutralize the freeacids in 1 gram of sample. With samples that contain virtually no free acids other than fatty acids, theacid value may be directly converted by means of a suitable factor to percent free fatty acids.SCOPEApplicable to crude and refined animal, vegetable and marine fats and oils, and various productsderived from them. Formerly Cd 3a-63.

APPARATUS1. Erlenmeyer flasks250 or 300 mL.2. Magnetic stirring device.3. Burette10mL, graduated in 0.05-mL divisions with a

tip drawn to a fine opening and extending at least 10cm below the stopcock.

4. Analytical balanceaccurate to 0.0001 g.

REAGENTS1. Potassium hydroxide (KOH), 0.1 NReagent Grade

KOH having a carbonate specification of 0.5% Max, or0.1 N KOH with NIST traceable standardization to 1part in 1000 in water, methanol or ethanol.

2. Solvent mixture consisting of equal parts by volume ofisopropyl alcohol (AOCS Specification H 18-58) andtoluene (AOCS Specification H 19-58). See Notes,Caution. The mixture must give a distinct and sharpend point with phenolphthalein in the titration as notedin Procedure, 5.

3. Phenolphthalein indicator solution1.0% in isopropylalcohol.

PROCEDURE1. Add indicator solution to the required amount of

solvent in ratio of 2 mL to 125 mL and neutralize withalkali to a faint but permanent pink color.

2. Determine the sample size from the following table:

Mass, g of sample Weighing accuracy,Acid value (10%), g g

01 20 0.0514 10 0.02415 2.5 0.01

1575 0.5 0.00175 and over 0.1 0.0002

3. Weigh the specified amount of a well-mixed liquidsample into an Erlenmeyer flask.

4. Add 125 mL of the neutralized solvent mixture. Be surethat the sample is completely dissolved before titrating.Warming may be necessary in some cases.

5. Shake the sample vigorously while titrating with stan-dard alkali to the first permanent pink color of thesame intensity as that of the neutralized solvent beforethe latter was added to the sample. The color mustpersist for 30 s.

6. Perform a blank titration using 125 mL of the neutral-ized solvent mixture.

CALCULATIONS1. Acid value, mg KOH/g of sample =

(A B) N 56.1W

WhereA = volume, mL of standard alkali used in the

titrationB = volume, mL of standard alkali used in titrat-

ing the blankN = normality of standard alkaliW = mass, grams of sample

To express in terms of free fatty acids as percent lauric,oleic, or palmitic, divide the acid value by 1.99, 2.81 or2.56, respectively.

PRECISION1. Single determinations performed in two different labo-

ratories should not differ by more than 0.22 for valuesless than 4 nor by more than 0.36 for values in therange 420.

ALTERNATE PROCEDURE FOR HIGHLY COLOREDSAMPLES

APPARATUS1. Glass electrodecalomel electrode pH meter for elec-

trometric titration. A sleeve-type calomel electrodeshould be used (see Notes, 2).

2. Variable-speed mechanical stirrerwith glass stirringpaddle.

3. Burette10 mL, graduated in 0.05-mL divisions with atip drawn to a fine opening and extending at least 10cm below the stopcock.

4. Beakers250 mL.5. Stand and mountings for electrodes, stirrer and burette.6. Magnetic stirring device.

REAGENTS1. Same as for the phenolphthalein titrimetric procedure,

except that the standard alkali should be standardizedby electrometric titration of pure potassium acid phtha-late and no indicator solution is necessary.

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PROCEDURE1. Determine the sample size from Procedure, 2 and

weigh the sample into a 250-mL beaker.2. Add 125 mL of solvent mixture.3. Mount the beaker in the titration assembly so that the

electrodes are half immersed. Start the stirrer and oper-ate at speeds that will give vigorous agitation withoutsplattering. Immerse the tip of the burette to 1 cmbelow the surface of the sample.

4. Titrate with suitable increments of alkali. After eachaddition of alkali, wait until the meter reading is essen-tially constant (usually within 2 min), then recordburette and meter readings graphically. Limit incre-ments of alkali so that changes in meter readings are 0.5pH units (0.03 volts) or less; when inflections in titra-tion curve occur, add alkali in 0.05-mL portions.

5. Remove titrated solution, rinse electrodes withisopropyl alcohol and immerse in distilled water.

6. Pe r fo rm a b lank t i t r a t ion , us ing 125 mL ofsolvent mixture.

CALCULATIONS1. Acid value, mg KOH/g of sample =

(A B) N 56.1W

WhereA = volume, mL of standard alkali used in titrat-

ing to middle of inflection in titration curveof sample

B = volume, mL of standard alkali used in titrat-ing to same pH meter reading for the blank

N = normality of standard alkaliW = mass, grams of sample

To express in terms of free fatty acids as percent oleic,lauric or palmitic, divide the acid value by 1.99, 2.81or 2.19, respectively.

NOTESCautionIsopropyl alcohol is flammable and a dangerous fire risk.The explosive limits in air are 212%. It is toxic by inges-tion and inhalation. The TLV in air is 400 ppm.

Toluene is flammable and a dangerous fire risk.Explosive limits in air are 1.277%. It is toxic by ingestion,inhalation and skin absorption. The TLV is 100 ppm in air.A fume hood should be used at all times when usingtoluene.

NUMBERED NOTES1. A standard methanolic potassium hydroxide (0.1 N)

solution (see AOCS Specification H 15-52) may beused as an alternate titrant in place of the standardaqueous solution. The methanolic potassium hydroxideis reported to provide a complete solvent system,having a distinct, clear end point. See References, 1regarding the ruggedness of the acid value method.

2. The pH meter should be standardized to pH 4.0 withstandard buffer solution. Immediately before using,wipe the electrodes thoroughly with clean cloth ortissue and soak for several minutes in distilled water.At weekly intervals, or more often if necessary, cleanthe glass electrode in a suitable cleaning solution.Also, drain calomel electrode and refill with freshpotassium chloride (KCl) electrolyte at weekly inter-vals. Both electrodes should be stored in distilled waterwhen not in use.

REFERENCES1. J. Assoc. Off. Anal. Chem. 59:658 (1976).

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Cd 3d-63 Acid Value

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AOCS Official Method Ca 12-55Replaced Ca 12-53 Reapproved 1997

PhosphorusDEFINITIONThis method determines phosphorus or the equivalent phosphatide content by ashing the sample inthe presence of zinc oxide, followed by the spectrophotometric measurement of phosphorus as a bluephosphomolybdic acid complex.SCOPEApplicable to crude, degummed and refined vegetable oils.

APPARATUS1 . Vycor cru c i bles, or equiva l e n t c ap able of withstanding

a mu ffle furnace temperat u re of at least 600C, 50 mL.2 . Wat ch glasses75-mm diameter.3 . E l e c t ric hot platewith rheostat contro l .4 . M u ffle furnacewith py rometer and suitable contro l s

to maintain a temperat u re of 550600C.5 . Funnelglass, short stem, 50-mm diameter.6 . Filter paperashless, 90-mm diameter, Wh atman no.

42, or equiva l e n t .7 . Wash bottle1 liter, for use with hot wat e r.8 . Vo l u m e t ric flasks50, 100, 250 and 500 mL, with

glass stoppers .9 . Tra n s fer pipets2, 5, 10 and 25 mL.

1 0 . PipetMohr type, 10 mL, with 0.1-mL subdiv i s i o n s .1 1 . S p e c t rophotometermeeting the re q u i rements of

AOCS Official Method Cc 13c-50, at a minimum, cap a-ble of measuring absorbance at 650 nm with a rep e at a-bility of 0.5%.

1 2 . C u vettes1.000 0.005 cm, for use in the visibl eregion of the spectru m .

REAGENTS1 . H y d ro ch l o ric acid (HCl)concentrat e d, sp gr 1.19 (see

Notes, C a u t i o n) .2 . Zinc ox i d e c o m m e rcial grade or re agent gra d e.3 . Potassium hy d roxide (KOH) pelletsre agent gra d e

(see Notes, C a u t i o n) .4 . S u l f u ric acid (H2S O4) c o n c e n t rat e d, sp gr 1.84 (see

Notes, C a u t i o n) .5 . Sodium moly b d at e re agent gra d e.6 . H y d razine sulfat e re agent grade (see Notes, C a u t i o n) .7 . Potassium dihy d rogen phosphat e re agent gra d e, dri e d

at 101C for 2 hr befo re use.

SOLUTIONS1 . Sodium moly b d at e C a re f u l ly add 140 mL of concen-

t rated H2S O4 to 300 mL of distilled wat e r. Cool toroom temperat u re and add 12.5 g of sodium moly b d at e.Dilute to 500 mL with distilled wat e r, mix thoro u g h lyand allow the solution to stand for at least 24 hr befo reu s e.

2 . H y d razine sulfat e, 0.015%Dissolve 0.150 g ofhy d razine sulfate in 1 liter of distilled wat e r.

3 . Potassium hy d rox i d e, 50% solutionDissolve 50 g ofKOH in 50 mL of distilled water with cooling (seeNotes, C a u t i o n) .

4 . S t a n d a rd phosphate solution(a) S t a n d a rd stock solutionprep a red by dissolving

1.0967 g of dry potassium dihy d rogen phosphat e(Reagents, 7) in distilled water and diluting to 250mL in a volumetric flask. This solution contains 1mg of phosphorus per mL.

(b) Standard working solutionprepared by pipetting5 mL of the standard stock solution into a 500-mLvo l u m e t ric flask and diluting to volume withdistilled water. This solution contains 0.01 mg ofphosphorus per mL.

PROCEDURE1 . Weigh 3.03.2 0.001 g of sample into a Vycor

c ru c i bl e. Add 0.5 g of zinc ox i d e.2 . H e at slow ly on a hot plate until the sample thicke n s ,

then gra d u a l ly increase the heat until the mass isc o m p l e t e ly ch a rre d.

3 . Place the cru c i ble in a mu ffle furnace at 550600C andhold for 2 hr. Remove and cool to room temperat u re.

4 . A dd 5 mL of distilled water and 5 mL of concentrat e dHCl to the ash.

5 . C over the cru c i ble with a wat ch glass and heat to ge n t l eboil for 5 min.

6 . Filter the solution into a 100-mL vo l u m e t ric fl a s k .Wash the inside of the wat ch glass and the sides of thec ru c i ble with about 5 mL of hot distilled wat e r, using awash bottle with a fine stream of wat e r. Wash thec ru c i ble and the filter paper with four additional 5-mLp o rtions of hot distilled wat e r.

7 . Cool the solution to room temperat u re and neutra l i ze toa faint turbidity by the dropwise addition of 50% KO Hsolution. Add concentrated HCl dropwise until the zincoxide pre c i p i t ate is just dissolve d, then add 2 add i t i o n a ld rops. Dilute to volume with distilled water and mixt h o ro u g h ly.

8 . Pipet 10 mL of this solution into a clean, dry 50-mLvo l u m e t ric flask (see Notes, 1).

9 . A dd 8.0 mL of hy d razine sulfate solution and 2.0 mL ofsodium moly b d ate solution, in the order give n .

1 0 . Stopper and inve rt three or four times. Loosen the stop-per and heat for 10 0.5 min in a vigo ro u s ly boilingwater bat h .

1 1 . R e m ove from the bath, cool to 25 5C in a water bat h ,dilute to volume and mix thoro u g h ly (see Notes, 2).

1 2 . Tra n s fer the solution to a clean, dry cuve t t e. Measurethe absorbance at 650 nm with the instrument adjusted

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to read 0% absorbance (100% transmittance) for ac u vette containing distilled water (see Notes, 1).

1 3 . P rep a re a re agent blank with no added oil by fo l l ow i n gthe pro c e d u re as described in Pro c e d u re, 112.

1 4 . M e a s u re the phosphorus content of the sample and theblank by comparison with the standard curve, obtainedas fo l l ow s :Preparation of Standard Curve(a) Pipet 0.0, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mL of the

s t a n d a rd wo rking solution [Solutions, 4, (b)] into50-mL vo l u m e t ric flasks. Dilute each to 10 mLwith distilled wat e r, using a measuring pipet.P roceed as directed in Pro c e d u re, 912. Th ealiquots re s p o n d, re s p e c t ive ly, to 0.0, 0.01, 0.02,0.04, 0.06, 0.08 and 0.10 mg of phosphorus.

(b) Plot the absorbance of each standard against itsphosphorus content in milligrams on linear graphpaper.

CALCULATIONS10 (A B)

1 . P h o s p h o rus, % =W V

WhereA = phosphorus content of sample aliquot in mgB = phosphorus content of blank aliquot in mgW = mass of sample in gV = volume of aliquot taken in Procedure, 8.

2 . E q u ivalent phosphatides, % = % phosphorus 30 (seeNotes, 4)

NOTESCautionH y d ro ch l o ric acid is a strong acid and will cause seve rebu rns. Pro t e c t ive clothing should be wo rn when wo rk i n gwith this acid. It is toxic by ingestion and inhalation and astrong irritant to eyes and skin. The use of a properly oper-ating fume hood is recommended. When diluting the acid,always add the acid to the water, never the reverse.

Potassium hy d rox i d e, like all alkalies, can bu rn skin,eyes and re s p i rat o ry tract seve re ly. Wear heavy ru bb e rgloves and face shield to protect against concentrated alkaliliquids. Use effective fume-removal device or gas mask top rotect re s p i rat o ry tract against alkali dusts or vap o rs .

When wo rking with ex t re m e ly caustic mat e rials, such aspotassium hy d rox i d e, always add pellets to water and notthe reverse. Alkalies are extremely exothermic when mixedwith water. Take precautions to contain the caustic solutionin the event that the mixing container breaks from theextreme heat generated.

S u l f u ric acid is a strong acid and will cause seve rebu rns. Pro t e c t ive clothing should be wo rn when wo rk i n gwith this acid. It is an oxidizing agent and should not bestored in the vicinity of organic materials. Use great cautionin mixing with water due to heat evolution that can causeexplosive splattering. Always add the acid to water, neverthe reverse.

H y d razine sulfate can cause eye, skin and mucous mem-b rane irri t ation, and liver and kidney damage. This com-pound is a known carc i n ogen in lab o rat o ry animals, causinglung and liver tumors in rats. It is a suspected human car-c i n ogen. Precautions in handling this compound include useof gloves, eye protection and re s p i rat o ry protection. Avo i dthe inhalation of dust and pow d e r. Dispose of waste mat e ri a land waste solutions in a proper and safe manner.

NUMBERED NOTES1 . If the absorbance of the colored solution measured in

P ro c e d u re, 12 is too high (>0.9), take a smaller sizealiquot of the sample solution than specified inP ro c e d u re, 8 (e. g., 2.0 mL), dilute to 10 mL withdistilled water using a measuring pipet and continue asd i rected in Pro c e d u re, 912.

2 . Samples of high phosphorus content may still giveabsorbencies >0.9. If this is the case, pipet 10 mL of thesample solution prep a red in Pro c e d u re, 7 into a 100-mLvo l u m e t ric flask and dilute to volume with distilledwat e r. Carry out the color development sequence notedin 812, using an ap p ro p ri ate size aliquot diluted to 10mL with distilled wat e r. Multiply the phosphoru scontent obtained, using the equation in Calculations, bythe dilution factor (10, if fo l l owing the pro c e d u red e s c ribed in this paragrap h ) .

3 . No unre a s o n able delay should be incurred betwe e nd eveloping the color in Pro c e d u re, 11 and measuri n gthe absorbance in Pro c e d u re, 14.

4 . This is an ap p rox i m ation for conve rting percent phos-p h o rus to percent phosphatides in soybean oil.

Page 2 of 2


Ca 12-55 Phosphorus

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AOCS Official Method Ca 12a-02Approved 2002

Colorimetric Determination of PhosphorusContent in Fats and Oils

DEFINITIONThe test portion is carbonized in the presence of magnesium hydroxycarbonate and then ashed. Theash is dissolved in dilute hydrochloric acid after which the phosphorus is determined colorimetricallyby the molybdenum blue method.SCOPEThis method specifies a colorimetric method for the determination of the phosphorus content of ani-mal and vegetable oils and fats (see Notes, 1).

APPARATUSUsual laboratory apparatus and, in particular, the following:

1. Test tubes, of 25-mL capacity, made of borosilicateglass, with stoppers and standard tapered necks, andgraduated at 5 mL-intervals or less. The reproducibilityof the 15 mL-graduation should be checked, as well asthe resistance of the calibration marks to heating at550C.

2. Block or muffle furnace, thermostatically controlledfor temperatures up to 400C.

3. Ashing oven or muffle furnace, suitable for tempera-tures up to 700C.

4. Tube rack, resistant to high temperatures, preferably ofcorrosion resistant steel, for use in the muffle furnace.The rack should hold the tubes at such an angle that theopen ends are about 3 cm above the bottom of thetubes.

5. Spectrophotometer, suitable for measurements at 720nm, using 1-cm and 4-cm cells.

6. Spectrophotometer cells, of path length 1 cm, and 4cm, and suitable for measurements at 720 nm.

REAGENTSUse only reagents of recognized analytical grade, unlessotherwise stated.

1. Magnesium hydroxycarbonate[(MgCO3)nMg(OH)2]H2O with a MgO content of 40% to 46% (by mass).Magnesium carbonate, hydrated, basic, [(MgCO3)4Mg(OH)2]5H2O is suitable.

2. Hydrochloric acidc(HCl) = 2 mol/L.3. Sodium hydroxide solutionc(NaOH) = 5 mol/L.4. Reducing solutionWeigh out 0.500 g metol

[(HOC6H4NHCH3)2H2SO4], 2.5 g sodium bisulfiteheptahydrate (Na2SO37H2O) and 58.5 g sodiummetabisulfite (Na2S2O5). Transfer the weighed outmaterials to a 1-L volumetric flask, dissolve in water,make up to the mark, and mix. Keep the solution in awell-sealed brown bottle.

5. Sulfate-molybdate reagentDissolve 25.0 g of ammo-nium molybdate tetrahydrate [(NH4)6Mo7O244H2O]in 250 mL of sulfuric acid solution c(H2SO4) = 5mol/L (prepared by diluting 278 mL sulfuric acid,c(H2SO4 = 18 mol/L, with water to 1 liter). Transfer

the solution to a 1-L volumetric flask, make up to themark with water, and mix. Store the solution in abrown bottle. WARNING: Care must be taken whendiluting concentrated sulfuric acid.

6. Sodium acetate solutionDissolve 340 g of sodiumacetate trihydrate (CH3COONa3H2O) in water, trans-fer to a 1-L volumetric flask, make up to the mark withwater, and mix. Store the solution in a brown bottle.

7. Standard phosphate solution for calibrationa. Stock solution (phosphorus content ca 100

g/mL)Weigh to 0.1 mg, about 440 mg ofpotassium dihydrogen phosphate (KH2PO4).Dissolve it in water, transfer quantitatively to a 1-L volumetric flask, make up to the mark withwater, and mix. Calculate the phosphorus contentof the solution by the formula:

ms MpPb =

V Ms

WherePb = the phosphorus content of stock solution in

micrograms per milliliter (g/mL)ms = the mass of potassium dihydrogen phosphate in

milligramsMp = the molar mass of phosphorus in grams (Mp =

31.03 g)V = the volume of stock solution in flask in liters (V =

1)Ms = the molar mass of potassium dihydrogen phos-

phate in grams (Ms = 136.09 g)

b. Standard phosphate solution 1, (phosphorus con-tent ca 10 g/mL). Pipette 25 mL of stock solution[Reagents, 7(b)] into a 250-mL volumetric flask,make up to the mark with water, and mix.Calculate the phosphorus content of this solutionby the formula:

Ps1 = 0.1 Pb

WherePs1 = the phosphorus content of standard phosphate

solution 1, in micrograms per milliliter (g/mL)

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Pb = the phosphorus content of the stock solution[stock solution (phosphorus content ca 100g/mL)] in micrograms per milliliter (g/mL)

c. Standard phosphate solution 2, (phosphorus con-tent ca 50 g/mL). Pipette 50 mL of stock solution(Reagents, 1) into a 100 mL volumetric flask,make up to the mark with water and mix.Calculate the phosphorus content of this solutionby the formula:

Ps2 = 0.1 Pb

WherePs2 = the phosphorus content of standard phosphate

solution 2, in micrograms per milliliter (g/mL)Pb = the phosphorus content of the stock solution

[Reagents, 7(a)] in micrograms per milliliter(g/mL)

SAMPLINGIt is important that the laboratory receive a sample which istruly representative and has not been damaged or changedduring transport or storage. A recommended samplingmethod is given in AOCS Official Method C 1-47 or ISO5555. Store samples in glass or polyethylene terephthalate(PET) bottles.

PREPARATION OF SAMPLEIf the sample is not completely molten at room tempera-ture, heat it to a maximum of 10C above the melting point.If the sample is not clear when liquid, homogenize it care-fully immediately before weighing out the test portions. Itis essential that any sediment, which may be rich in phos-phorus, is incorporated homogeneously into the sample.

PROCEDURE1. Determination of the calibration factor

a. For phosphorus contents of 0 mg/kg to 125 mg/kg(in the oil)1. Weigh 30 mg of magnesium hydroxycarbon-

ate (Reagents, 1) into each of a series of 7 testtubes (Apparatus, 1), and using a microburetteor pipette add to the test tubes 0 mL (blank);0.25 mL; 0.5 mL; 1.0 mL; 1.5 mL; 2.0 mLand 2.5 mL of standard phosphate solution 1[Reagents, 7(b)] so that the test tubes containquantities of phosphorus ranging from 0 g toca 25 g, equivalent to the amount of phos-phorus in 0.2 g of an oil containing between 0mg/kg to about 125 mg/kg phosphorus (seeNotes, 2).

2. Add 2 mL of hydrochloric acid (Reagents, 2)to each test tube and wait until a clear solutionis obtained. Then add 0.5 mL of sodiumhydroxide solution (Reagents, 3) to each testtube and mix.

3. Using a pipette or burette add 5 mL of reduc-ing solution (Reagents, 4) to each test tubeand mix.

4. In a similar manner add 2.5 mL of sulfate-molybdate reagent (Reagents, 5) to each testtube and mix. Stopper the tubes and let themstand for 20 minutes in a dark place.

5. Fill the test tubes to the 15 mL mark with sodi-um acetate solution (Reagents, 6) and mix.

6. Measure the absorbance of the solutionsagainst the blank, in a 4-cm cell, at 720 nm(see Notes, 3).

7. Calculate the calibration factor in accordancewith Procedure 1(c). Alternatively compute theequation of the regression line for the calibration.

b. For phosphorus contents of 125 mg/kg to 500mg/kg (in the oil)1. Weigh 30 mg of magnesium hydroxycarbon-

ate (Reagents, 1) into each of a series of 6 testtubes, and using a microburette or pipette addto the test tubes 0 mL (blank); 0.5 mL; 0.8mL; 1.2 mL; 1.6 mL and 2.0 mL of standardphosphate solution 2 [Reagents, 7 (c); phos-phorus content ca 50 g/mL]. so that the testtubes contain quantities of phosphorus rang-ing from about 25 g to 100 g, equivalent tophosphorus contents of about 125 mg/kg to500 mg/kg in the oil. The absorbance of thehighest standard, in a 1-cm cell, should beapproximately 0.8.

2. Add 2 mL of hydrochloric acid (Reagents, 2)to each test tube and wait until a clear solutionis obtained. Then add 0.6 mL of sodiumhydroxide solution (Reagents, 3) to each testtube and mix.

3. Using a pipette or burette add 5 mL of reducingsolution (Reagents, 4) to each test tube and mix.

4. In a similar manner, add 2.5 mL of sulfate-molybdate reagent (Reagents, 5) to each testtube and mix. Stopper the test tubes and letthem stand for 20 minutes in a dark place.

5. Fill the test tubes to the 15 mL mark with sodi-um acetate solution (Reagents, 6) and mix.

6. Measure the absorbance of the solutionsagainst the blank, in a 1-cm cell, at 720 nm(see Notes, 3).

7. Calculate the calibration factor in accordancewith Procedure, 1 (c). Alternatively compute theequation of the regression line for the calibration.

c. Calculation of the calibration factor: For eachsolution i of the series measured for phosphoruscontents of 0 mg/kg to 125 mg/kg (in the oil) andfor phosphorus contents of 125 mg/kg to 500mg/kg (in the oil) calculate the calibration factorusing the formula:

(Vi Ps)fi = AiWhere

fi = the calibration factor for solution i of the series,in micrograms

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Ca 12a-02 Colorimetric Determination of Phosphorus Content in Fats and Oils

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Vi = the volume of standard phosphate solution insolution i, in millilitres

Ps = the phosphorus content of the phosphate solutionused, in micrograms per milliliters (g/mL)

Ai = the absorbance measured for solution i

Use the average of the factors fi as the calibrationfactor f for the calculation in (Calculation).

2. Ashing of oil samplea. Weigh approximately 30 mg of magnesium

hydroxycarbonate (Reagents, 1) into a test tube(Apparatus, 1) and weigh the test tube, with themagnesium hydroxycarbonate, to 0.1 mg.

b. Using a Pasteur pipette add approximately 0.2 g(10 to 15 drops) of the oil sample (6), taking carethat all the sample material falls to the bottom ofthe tube and mixes with the magnesium hydroxy-carbonate. No drops should be allowed to fall orsplash onto the side walls of the test tube.

c. Re-weigh the test tube to 0.1 mg. d. Prepare a blank test tube containing magnesium

hydroxycarbonate only. e. Place the test tubes in the heating block or in the

tube rack (Apparatus, 4) in the cold muffle furnace(Apparatus, 2). Heat the test tubes to 350C untilthe sample is carbonized to a dry black mass (1hour to 2 hours).

f. After carbonization, increase the temperature to550C and heat the sample at this temperatureuntil the ash is completely white (about 2 hours).

g. Remove the test tubes (and tube rack) and allowthe test tubes to cool.

3. Colorimetric determinationa. Dissolve the residue from the ashing procedure in

2 mL of hydrochloric acid (Reagents, 2) by warm-ing carefully until the liquid boils.

b. Allow the test tubes to cool and neutralize the con-tents by adding 0.6 mL of sodium hydroxide solu-tion (Reagents, 3) to each, then add, using a mea-suring pipette or burette, 5 mL of reducingsolution (Reagents, 4) and mix.

c. In a similar manner add 2.5 mL of sulfate-molyb-date reagent (Reagents, 5) and mix.

d. Stopper the test tubes and allow them to stand in adark place for 20 minutes.

e. Fill the test tubes to the 15 mL mark with sodiumacetate solution (Reagents, 6) and mix.

f. Measure the absorbance of the solution against theblank in a 4-cm cell at 720 nm (see Notes, 6).

g. If the measure of absorbance is higher than that ofthe highest standard (about 0.8) it lies outside thecalibration range and the measurements must berepeated, using a 1 cm cell.

CALCULATIONCalculate the phosphorus content using the formula:

(f A)wp = m

Wherewp = the phosphorus content of the sample, in mil-

ligrams per kilograms (mg/kg)f = the average calibration factor calculated as in

Procedure, 1 (c), in microgramsA = the absorbance measured as in Reagents, 3m = is the mass of the sample, in grams

Alternatively, if the equation of a regression line is used forthe calculation of the phosphorus content of the test portionthen:

mpwp = m

Wherewp = the phosphorus content of the oil, in milligrams

per kilograms (mg/kg)mp = the phosphorus content of the test portion

(Reagents, 2), in microgramsm = the mass of the test portion (Reagents, 2), in

grams

PRECISION1. Interlaboratory testDetails of interlaboratory tests on

the precision of the method are summarized in Table 1.The values derived from these interlaboratory testsmay not be applicable to concentration ranges andmatrices other than those given. Two international testswere carried out on samples of soybean oil using thecolorimetric method.a. Results of the first interlaboratory test (1995)

The first interlaboratory test was organized by theFederation of Oils, Seeds and Fats AssociationsLtd. (FOSFA) involving 8 laboratories in 4 coun-tries. It was carried out in July 1995. The resultsobtained were subjected to statistical analysis inaccordance with ISO 5725:1986 (3); the precisiondata are shown in Table 1.

b. Results of the second interlaboratory test (1999)The second interlaboratory test was organized byFOSFA International and the American Oil

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Table 1Statistical results of the first interlaboratory test (1995).

Samplea

Parameter A B

Number of participating laboratories after eliminating outliers 8 8

Mean value, mg/kg 10.51 318.31Repeatability standard deviation, sr, mg/kg 0.68 4.41Coefficient of variation of repeatability, % 6.47 1.39Repeatability limit r (r = 2.8 sr), mg/kg 1.92 12.34Reproducibility standard deviation, sR, mg/kg 2.12 17.93Coefficient of variation of reproducibility, % 20.17 5.63Reproducibility limit R (R = 2.8 sR), mg/kg 5.96 50.21

aSample A: RBD soybean oil.Sample B: Crude water degummed soybean oil.

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Chemists Society (AOCS), involving 13 laborato-ries. The results obtained were subjected to statis-tical analysis in accordance with ISO 5725-1:1994and ISO 5725-2:1994; the precision data areshown in Table 2.

The same samples were also tested usingatomic absorption spectrometry (AAS), and induc-tively coupled plasma (ICP) optical emission spec-trometry. Statistical results related to AAS andICP methods are presented in Ca 12b-92 and Ca20-99, respectively.

2. RepeatabilityThe absolute difference between twoindependent single test results, obtained using the samemethod on identical test material in the same laborato-ry by the same operator using the same equipmentwithin a short interval of time will in not more that 5%of cases be greater than the repeatability limit (r),deduced by linear interpolation from Table 3.

3. ReproducibilityWhen the values of two single testresults, obtained using the same method on identicaltest material in different laboratories with differentoperators using different equipment, lie within therange of the values in Table 3, the absolute differencebetween the two test results will in not more than 5%of cases be greater than the reproducibility limit (R),deduced by linear interpolation from Table 3.

NUMBERED NOTES1. This method is not suitable for determining the phos-

phorus content of commercial lecithin as this requiresan ashing temperature of 800C.

2. The absorbance of the highest standard, containing 2.5mL of standard phosphate solution 1, under these con-ditions should be approximately 0.8.

3. Alternatively, measure the absorbance of all the solu-tions against water, as a check on the blank, and then,unless the equation of a regression line is to be comput-ed, correct all the measurements for the blank value.

REFERENCES1. ISO 5555:1991, Animal and vegetable fats and oils

Sampling.2. ISO 661:1989, Animal and vegetable fats and oils

Preparation of test samples.3. ISO 5725:1986, Precision of test methods

Determination of repeatability and reproducibility for astandard test method by interlaboratory tests (with-drawn in 1994).

4. ISO 5725-1:1994, Accuracy (trueness and precision) ofmeasurement methods and resultsPart 1: Generalprinciples and definitions.

5. ISO 5725-2:1994, Accuracy (trueness and precision )of measurement methods and resultsPart 2: Basicmethod for the determination of repeatability andreproducibility of a standard measurement method.

6. ISO/DIS 10540-2:2001, Animal and vegetable fats andoilsDetermination of phosphorus contentPart 2:Method using graphite furnace atomic absorption spec-trometry.

7. ISO/DIS 10540-3:2001, Animal and vegetable fats andoilsDetermination of phosphorus contentPart 3:Method using inductively coupled plasma (ICP) opti-cal emission spectroscopy.

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Table 2Results of the second interlaboratory test (1999).

Samplea

Parameter A B C D E F G H

Number of participating laboratories after eliminating outliers 7 7 7 7 7 7 7 7Mean value, mg/kg 473.1 305.0 131.6 101.4 56.6 32.0 13.8 273.8Repeatability standard deviation, sr, mg/kg 14.2 5.1 3.5 4.3 3.0 2.8 1.1 2.4Coefficient of variation of repeatability, % 3.0 1.7 2.7 4.2 5.3 8.6 8.2 2.9Repeatability limit r (r = 2.8 sr), mg/kg 39.8 14.3 9.8 12.0 8.4 7.9 3.2 6.7Reproducibility standard deviation, sR, mg/kg 56.4 59.2 31.0 14.6 6.5 3.3 2.2 56.4Coefficient of variation of reproducibility, % 11.9 19.4 23.6 14.4 11.5 10.4 16.2 20.7Reproducibility limit R (R = 2.8 sR), g/kg 158.0 165.7 86.8 40.9 18.2 9.3 6.3 158.0

aSamples of a soybean oil containing different levels of phosphorus.

Table 3Repeatability and reproducibility limits at different phosphorus contents.

Phosphorus content r R

mg/kg mg/kg mg/kg10 2 650 8 18100 12 41300 13 105400 34 135

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AOCS Recommended Practice Ca 19-86Reapproved 1997

Phospholipids in Vegetable Oils Nephelometric Method

DEFINITIONThe nephelometric method measures turbidity in oilacetone mixtures due to phospholipids. The tur-bidity is correlated to the phosphorus level.SCOPEThis method is ap p l i c able to cru d e, deg u m m e d, once-re fi n e d, bl e a ched and deodori zed vege t abl eoils. The presence of soap in oil samples may give erroneous results (see Notes, 1).

APPARATUS1 . L ab turbidimetermodel 2100N (Hach Company,

L ove l a n d, CO, USA, 800-227-4224), or equiva l e n t .2 . Turbidimeter sample cells2.5 9.6 cm (Hach cat a l og

no. 20849-00), or equiva l e n t .3 . G e l ex secondary turb standard set. Hach cat a l og no.

2 2 5 - 2 6 - 0 0 .4 . M i c rowave oven or hot plat e.5 . Fluted filter paper12 cm, Wh atman no. 2, or equiva-

l e n t .6 . Filter funnel6-cm diameter.7 . P y rex beake rs150 mL.8 . Vo l u m e t ric flasks50 mL.9 . S t ablcal Calibration KitHach cat a l og no. 26621-00.

REAGENTS1 . A c e t o n e s p e c t ral grade (see Notes, C a u t i o n) .

PROCEDURE1 . Tu rn on the power sw i t ch of the turbidimeter and allow

it to wa rm up at least 15 min befo re use.2 . C a l i b rate the turbidimeter according to the manu fa c-

t u re r s instructions, and perfo rm a blank determ i n at i o non the acetone (see Notes, 2).

3 . H e at the oil sample to about 50C, using a microwaveoven or hot plate (see Notes, 3).

4 . Pass the sample through dry, fluted filter pap e r.5 . Weigh (see Notes, 4) an ap p ro p ri ate amount of the oil

sample (0.33, 1.67 or 8.35 g, depending on the phos-p h o rus level and the oil type) into a 50-mL vo l u m e t ri cfl a s k .

6 . A dd acetone to the 50-mL mark .7 . S t o p p e r, mix well and pour the mixed sample into the

t u r b i d i m e t ric cell (see Notes, 5) to the re q u i red leve l( about 30 mL).

8 . C ap the sample cell and shake by hand for about 10 sec.9 . Wipe the sample cell clean with a tissue and place it in

the turbidimeter (see Notes, 6 and 7).1 0 . Select the correct turbidity ra n ge: either 2, 20 or 200

n ep h e l o m e t ric turbidity units (NTU).1 1 . R e c o rd the turbidity reading after ex a c t ly 5 min (see

Notes, 8).1 2 . S u b t ract the NTU value for the acetone blank from the

reading obtained in step 11.

NEPHELOMETRIC PHOSPHORUS DETERMINATION1 . The phosphorus level in mg/L (ppm) for a given oil

type can be determined nep h e l o m e t ri c a l ly either bye s t i m ating phosphorus dire c t ly from a phosphorus vs.NTU curve, or by calculating phosphorus from thec o rresponding equat i o n .

PREPARATION OF CORRELATION GRAPH ANDEQUATION FOR CURVE

1 . For a given type of vege t able oil, obtain ap p rox i m at e ly1015 samples of either cru d e, deg u m m e d, once-re fi n e d, bl e a ched or deodori zed oils.

2 . D e t e rmine the turbidity of each sample, using samples i zes indicated in Table 1. If the sample is diffe re n tf rom corn or soybean oil, sample size must be chosen togive NTU values similar to those shown in Table 1.

3 . D e t e rmine phosphorus level of each sample, usingAOCS Official Method Ca 12-55.

4 . Plot phosphorus in mg/L (ppm) vs. turbidity (NTUvalues) for each sample.

5 . C a l c u l ate the equation for the data set using least-s q u a res analy s i s .

Table 1Guide for measurement of phosphorus in soybean and cornoils at various levels of refinement.

RecommendedOil type Equation for curve sample size, g

SoybeanCrude P = (5.89 NTU) + 316.4 0.33Degummed P = (5.32 NTU) + 3.38 1.67Once-refined P = (8.26 NTU) 4.49 1.67Bleached P = (1.27 NTU) 0.225 8.35Deodorized P = (1.72 NTU) 0.528 8.35

CornCrude P = (5.62 NTU) + 97.2 0.33Degummed P = (3.69 NTU) 2.77 1.67Once-refined P = (1.42 NTU) 2.21 1.67Bleached P = (2.60 NTU) 1.05 8.35Deodorized P = (0.99 NTU) + 0.027 8.35

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EQUATIONS FOR CURVES AND SAMPLE SIZES1 . See Table 1.

CALCULATION EXAMPLE1 . For crude soybean oil, the best-fit equation is

P = (5.89 NTU) + 316.4Where

P = phosphorus, mg/L (ppm)NTU = turbidity

The phosphorus levels of the other oil types are calcu-lated similarly, except using the appropriate equation.

NOTES1 . High soap levels (50100 mg/L or gre ater) in vege t abl e

oil may give erroneous va l u e s .2 . The turbidity of accep t able acetone for this method is

0.5 NTU or less.3 . H e ating the oil sample promotes faster fi l t ration. Highly

hy d roge n ated oil samples may need heating in ex c e s s

of 50C to assure complete melting. Some oil samplesfi l t e red in the re fining process may not re q u i re furt h e rfi l t ration befo re analysis. Oil sample moisture leve lshould not exceed 0.5%.

4 . Samples should be weighed to the nearest 0.01 g fo rbest accura cy.

5 . The interior and ex t e rior of the sample cell and capmust be cleaned with only low-NTU acetone prior toe a ch analy s i s .

6 . The temperat u re of the oilacetone mixture prior toa n a lysis should be 25C.

7 . The sample cell must be pro p e rly aligned, according tothe manu fa c t u re r s instructions, when placed in thet u r b i d i m e t e r.

8 . If after 5 min the turbidity reading has not stab i l i ze d,rep e at the entire pro c e d u re.

REFERENCESJ. Am. Oil Chem. Soc. 63(5):667 (1986).

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Ca 19-86 Phospholipids in Vegetable OIls Nephelometric Method

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AOCS Official Method Ca 20-99Revised 2000

Analysis for Phosphorus in Oil by Inductively Coupled Plasma Optical

Emission SpectroscopyDEFINITIONThis method describes a procedure for the quantification of phosphorus in oil using inductively cou-pled plasma optical emission spectroscopy (ICP-OES).SCOPEDepending on the dilution solvent, nearly all forms of vegetable oils may be analyzedfrom crudeoil to degummed, refined, bleached, deodorized, and hardenedand nearly all types of lecithins andphosphatides. This procedure is suitable only when the elements are present in a solubilized form.When they are present as fine particles such as bleaching earth, catalysts, or rust, ICP-OES analysisresults in poor recovery due to nebulization and atomization problems. The only suitable non-ashingdirect method for these samples is graphite furnace AAS.

APPARATUS1. Inductively coupled plasma optical emission spectrom-

eter (General Notes, 1)2. Analytical balance (4-place accuracy)3. Oven4. Disposable plastic vials5. Disposable pipet tips6. Tilt table mixer7. Volumetric flasks

REAGENTS1. 1-Butanol2. Kerosene or xylene (General Notes, 2)3. Standard elements as organic soluble:

(a) SPEX, 203 Norcross Ave., Metuchen, NJ 08840 (b) Constan brand standards ava i l able from: A c c u -

Standard, 25 Science Park, New Haven, CT 06511 4. Base 20 oil or Base 75 oil from Accu-Standard may be

used to check the blank oil used and for the dilution ofthe standard solutions as needed.

PRINCIPLESolvent-diluted vegetable oils are analyzed for phosphorusby direct aspiration. Liquid samples are nebu l i zed and car-ried into the ex c i t ation source by a fl owing gas. Atoms areq u a n t i t ated by measuring the specific emission lines pro-duced by atoms decaying from high energy leve l s .

PROCEDURE1. SamplingAll samples, standards, and blanks are dilut-

ed 1:1 with 1-butanol (or ke rosene/xylene) to reduce theviscosity of the oil for better nebu l i z ation. 1-Butanol isp re fe rred because it has better moisture tolerance anda l l ows a higher fl ow rate with higher pre s s u re thanke rosene without putting out the torch. The higher fl owrate provides for improved detection limits. Th ei n c reased moisture tolerance permits the analysis ofc rude oils and lecithins without phase sep a rat i o n .

( a ) H a rdened fats are fi rst melted and mixed prior todilution. The diluted sample is then kept wa rm andm o n i t o red throughout the analysis to insure itremains in solution. The re l at ive maximum meltingpoint of hardened fats analy z able is 60C. Howeve rsome are more soluble in 1-butanol than others.

(b) 2.5 g (0.02 g) of sample is weighed into anautosampler tube and diluted with 2.5 g of 1-butanol (ke rosene or xylene) delive red from anautopipetor, capped and inverted 4050 times.

(c) Lecithins up to 100% AI (0.2 g) are diluted to 5.0 g with blank soybean oil and then to 10 g with1-butanol. The sample is then mixed on a tilt tablem i xer for one hour, then diluted 1:10 with 1:1blank oil/1-butanol (total dilution 1:250).

2. I n t e rnal standard If an internal standard is desire d(see Standardization below), it should be incorporatedas part of the dilution step above. Typically, the resul-tant dilution should contain 10 mg/kg yttrium. Th u s ,under the dilution sequence for sampling, Procedure 1,(a), the 2.5 g of 1-butanol added as diluent should con-tain 20 mg/kg yielding 10 mg/kg yttrium in the 1:1dilution to accomplish this. Operate your instru m e n taccording to the manufacturers directions and specifi-cations (see General Notes, 1).

3. The instrument is ignited and allowed to wa rm. It isprofiled on an internal Hg lamp.

4. B l a n k S t a n d a rd i z ation is perfo rmed fi rst on a bl a n koil (typically refined and bleached soybean or other oilwhich has been shown to be free of trace elements) andrun at the specified wavelength for phosphorus. Base20 or Base 75 oil is used as an absolute reference blankto determine that the blank oil used is free of elements.A blank oil is diluted 1:1 as described in the samplingprocedure and analyzed directly.

5 . S t a n d a rd i z ationA single standard, prep a red from ac o m m e rc i a l ly ava i l able single element orga n i c - b a s e ds t a n d a rd, is run at the phosphorus wavelength. Standard s

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AOCS Methods for Biodiesel Feedstock Quality