IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

PRINCIPLE
Dialkyl ketones can be formed by the wrong transesterification of the oil or fat and are
toxic substances and , therefore, are not desirable in food products.
The sample is saponified with ethanolic potassium hydroxide and then the unsaponifiable
portion (which contains dialkylketones) resulting is extracted with petroleum ether. The
petroleum ether is then washed and evaporated, and the residue which remains is
dissolved and analyzed by HPLC. Here, the separation takes place on a silica column,
after which the separated components are detected by a ELSD . Quantification
determined via calibration curve.

REAGENTS
1. 2N ethanolic KOH .
Add 35 grams of KOH in 25 ml of demineralized water and cool to room temperature.
After cooling, dilute with ethanol to the volume of 250 ml in the 250ml volumetric flask .
2. Washing solution : ethanol / demineralised water = 1 : 1 ( v / v ) .
Measure 1 part ethanol and 1 part distilled water and add them together .
3. Saturated sodium chloride solution .
Add a quantity of sodium chloride in a sample bottle of 100 ml and add distilled water
and shake vigorously . At the bottom of the container should be have excess sodium
chloride .

EQUIPMENT AND SUPPLIES

Requirements for sample preparation
1. For each sample, three glass vials with screw
2. 40 ml screw cap vials
3. 1000 ul Eppendorf pipette
4. Balance
5. Water bath filled with distilled water , adjusted to 90 C.
6. Pasteur pipette
7. Vortex
8. Vial of 25 ml
9. Vial 2 ml with screw cap and Teflon septum
10. Heating block
In addition, using standard laboratory equipment such as beakers and graduated
cylinders .
HPLC ELUENT
For HPLC, prepare the following solvents :

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD
Solution
Solution
Solution
Solution

A: Toluene
B : Hexane
C : Ethyl acetate
D: 3 % aqueous formic acid solution in toluene

Solution D is created as follows :

Add to 2.5 liter of toluene with 75 ml of formic acid or 1.0 liter of toluene with 30 ml of
formic acid.
Formic acid dissolves poorly in toluene , so shake vigorously until a homogeneous
mixture has emerged.
HPLC EQUIPMENT
1. ELSD detector (Varian 380-LC)
2. HPLC system suitable for ternary gradient
3. Silica column (3 uM , 150 * 4.6 mm)
4. Chromatographic data processing system (Ez Chrome)
The settings of the equipment.

HPLC DETECTOR SETTING

1. Turn on the detector.
2. Set the detector as follows :

Drift Tube temp : 75 C

Gas Flow : 1.75 SLPM
Always give as an auto zero

HPLC AUTOSAMPLER SETTING

Set the autosampler with the following settings :

Run Time: 25.5 minutes

Injection volume: 20 ul

HPLC PUMP SETTING

1. When the detector settings are reached , the pump will automatically turn on.

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

2. Adjust the flow rate of the pump at 0.90 ml / min.

3. The polar components of the column is built up with the solutions A , B, C and D.
For this, the HPLC pump must be programmed as follows :

4. The air bubbles should be removed by purge the solution approximately one
minute away in the suction.
5. When this is done, the solutions need to be in order to rinse the column.

6. The setup is now ready for analysis.

STANDARDS
1. DAK stock solution 250 mg / l .
Weigh 25 mg of pure DAK to 0.1 mg and place it into a 100 mL volumetric flask . Fill the
volumetric flask with hexane solution / toluene up to the mark. Record the weight of the
solvent.
2. DAk standard 1 50 mg / l .
Weigh the DAK stock solution 2 ml to 0.1 mg into a 10 mL volumetric flask and then fill it
with solution of hexane / toluene. Record the weight of the solvent.
3. DAK standard solution 2 10 mg / l.
Weigh the standard DAK 1 2 ml to 0.1 mg into a 10 mL volumetric flask and then fill it
with solution of hexane / toluene. Note the weight.
4. Hexane / toluene solution (1:1)

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD
*By noting the weight concentration of the two standard solutions can be calculated
exactly .

METHOD OF STANDARD PREPARATION

1. Security
When work-up the method, various chemicals are used. Take note of the specific hazards
of these chemicals. Make use of necessary personal protective equipment and work with
the use of a fume cupboard with window to the specified safe working height
2. Process for determining the calibration curve of the two standard solutions DAK and is
around 2 ml, with a Pasteur pipette in an HPLC vial pipetted. Then Put the two vials in
the carousel of the HPLC . Put the two standard blank.
3. Program the HPLC as follows :
STANDARD CONCENTRATION ( MG /
L)

INJECTION
( UL)

blank

VOLUME
20

10

20

10

40

10

60

10

80

50

20

50

40

50

60

50

80

4. Set the calibration sequence in the EzChrome as below :

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AUTOSAMPLER

IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

5. Start the HPLC.

From these data, a calibration curve made by comparing the mass DAK what is brought
into the column from the peak area. The DAK mass should be the exact concentration of
the two standard calculated. The R2 for the calibration curve must be >0.99.

SAMPLING AND PROCESSING

The samples should be preferably provided in clean sealable containers in amounts of at
least 10 grams. The quantity provides a representative sample and allows for re-analyze.
The sample for analysis should be clean and dry.
1. Liquid samples
The sample is homogenized by shaking it vigorously.
2. Solid samples
Solid samples are first gently melted in an oven (up to 70 C) or microwave oven until
form a homogeneous liquid . It is necessary to take into account the duration of the
heating up.

METHOD OF SAMPLE PREPARATION

1. Pipette into a test tube with an Eppendorf pipette 1000 ul oil or grease and weigh to
the nearest 1.0 g with a balance. Please record the weigh.
2. Add with a measuring cylinder 10 mL of 2N ethanolic KOH solution. Then add some
boiling granules. Close the test tube with a cap .
3. Put the tube sample for 20 minutes at 90 C in a water bath. It is imperative here
that the water bath is well filled with distilled water.
4. Cooling the test tube to room temperature. After cooling, add about 10 ml of distilled
water to the soap solution in the test tube . Homogenize the mixture. If the soap does
not dissolve , then heat the test tube for a few seconds in the water bath and
homogenize again .
5. Add with a graduated cylinder 5 ml of petroleum ether. Close the test tube with the
cap and shake the tube vigorously by hand for 1 minute.
6. Wait until two layers is formed in the test tube occur. The separation between the
layers can be accelerated by adding into the test tube a few drops of saturated
sodium chloride solution.
7. Pipette the petroleum ether layer in the second test tube with a Pasteur pipette . It s
alright if some of the water layer is pipetted along because the petroleum ether
extract still going to be washed .

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD
8. Repeat steps 5 until 7 another 2 more times.
9. Add with a graduated cylinder 10 ml of washing solution to the petroleum ether
extract in the second test tube . Close the test tube with the cap and shake the tube
vigorously by hand for 1 minute .
10. Leave the tube stand until two layers are visible in the test tube . Add a drop wise
saturated sodium chloride solution to accelerate the separation.
11. Pipette the petroleum ether layer in the third test tube . Again, its alright if some of
the water layer is pipetted along. Then repeat steps 9 until 10 another 1 times for the
third test tube .
12. Pipette the petroleum ether layer to a 25 ml vial with a Pasteur pipette . Make sure
that no water layer is suck together with the pipette .
13. Evaporate the petroleum ether under nitrogen down into the heating block.
14. Solve the remaining residue in 4 ml solution of hexane : toluene (1:1). Do this by
measure the hexane : toluene (1:1) solution in a graduated measuring cylinder.
15. Shake the vial using vortex for 1 minute.
16. Pipette Finally, about 2 ml of this solution with a Pasteur pipette in a 2 ml vial . The
sample is now ready to be injected.
17. Setting the HPLC equipment.
18. The sample info (sample amount, multiplier and dilutor) need to be insert in the
sequence.
19. Run the HPLC. The DAK content will be calculated automatically by the EzChrome.

SEQUENCE SETTING FOR THE SAMPLES

1. Sample info need to be insert in the EzChrome :

Method : DAK 2015

Sample amount : Sample weigh (g)
Multiplier : 4
Dilutor : 10

Example of sequence setting in the EzChrome :

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

CALCULATION AND REPORTING

1. Peak Identification

*In the chromatogram , the peak from the DAK is around 3.5 minutes.

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

* Example of chromatogram with no DAK content (Blank sample) :

*Example of chromatogram for sample that do not undergo any interesterification

process (IV64 sample) :

2. Quantification of the chromatogram

Peak areas are determined with the aid of the data processing system.

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD
3. Calculation
The peak area is obtained by means of the calibration curve converted to a given mass
DAK

DAK content

DAK mass 4
10 sample weight
Content
DAK
DAK
Concentration in the

=
sample

( mg / kg )
DAK mass

= Mass DAK

(ng)
Dissolution Volume = Volume in which sample is dissolved (ml )
Injection volume = Volume injected ( ul)
Sample weight
= Sample test portion weighed sample ( g )
4. Reporting
Report the final result. If more than one test is carried out on the same sample ,
report the average income and the number of repetitions . Round off results in an
integer.
5. Datastorage
All results are in the database put in the corresponding sample number

PRECISION OF THE METHOD

1. Repeatability
The repeatability is determined by a control sample, which had a DAK concentration of
mg / kg, and analyzed more than 10 times.
2. Reproducibility
The reproducibility was determined by analyzing the control sample on few different
days.

3. Special cases and comments

Validation:
Reproducibility is measured by different analysts on different days. This is done by using DAK control
standard from LCW and PH216E sample (this sample will be use as IOI LCA QC control standard).
LCW control standard (CLSP 555) :

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IOI LODERS CROKLAAN OILS SDN BHD

IOI LIPID ENZYMTEC SDN BHD

Mean
SD
Max
+2SD
+1SD
-1SD
-2SD
Min
N

666.27
25.25
713.68
716.77
691.52
641.03
615.78
635.04
15

PH216E sample (this sample will be use as IOI LCA QC control standard) :

Mean
SD
Max
+2SD
+1SD
-1SD
-2SD
Min
N

689.17
35.08
756.02
759.33
724.25
654.10
619.02
631.42
26

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