food application notes - develosil usa · 8. erythrosine b 9. phloxine b 10. rose bengal analytical...

Food Application Notes

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CONTENTSFood Application Notes

Six Common Acidic Compounds ..........................................................................................6

Fatty Acids ............................................................................................................................7

Ascorbic and Citric Acid in Drinks ........................................................................................8

Food Additives in a Soft Drink ............................................................................................11

Color Additives ....................................................................................................................13

Capsaicin ............................................................................................................................16

Capsaicin in Pepper Sauce .................................................................................................17

Capsaicin in Red Peppers ..................................................................................................18

Carotenes in Carrots ...........................................................................................................19

Catechins in Tea ..................................................................................................................20

Isoflavones ..........................................................................................................................23

Isothiocyanates ...................................................................................................................24

Maltooligosaccharides ........................................................................................................25

Preservatives .......................................................................................................................26

Resveratrol in Red Wine ......................................................................................................28

Sinigirin ...............................................................................................................................29

Saccharides ........................................................................................................................30

Oil Soluble Vitamins ............................................................................................................31

Water Soluble Vitamins .......................................................................................................32

Water Soluble Vitamins via Ion Pair Method .......................................................................36

Vitamin A .............................................................................................................................37

Vitamin D2 and Vitamin D3 ...................................................................................................38

Technical Report ......................................................................................................................39

Product Information .................................................................................................................43

Column Selection Guide .........................................................................................................46

About Us .................................................................................................................................48

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Separation of highly polar compounds by reverse phase column

True UHPLC realized with maximum column efficiency

• The strictly controlled particles are appropriately transferred to 5 µm, 3 µm, 2 µm.

• Ensure high durability by our unique bonding and filling technology.

• Maximized high column efficiency realizes low cost by reducing solvent usage

[Superior resolution and retention of HSR AQ C18 for UHPLC]

UHPLC method transfer of Purine Derivative

Conditions:

Column Develosil HSR AQ C18, 5 µm (4.6 x 150 mm) Develosil HSR AQ C18, 2 µm (2.0 x 50 mm)

Mobile Phase 0.1% TFA

Flow rate 1.0 mL/min (HPLC) 0.474 mL/min (UHPLC)

Temperature 30ºC

Detection UV220 nm

Sample

1. Allantoin

2. Hypoxanthine

3. Uric Acid

4. Xanthine

5. Inosine

Injection Volume2.0 uL (HPLC)

0.16 uL (UHPLC)

Gradient elution with organic solvent can further shorten it.Excellent isolation and retention will give great results to UHPLC method transfer.

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Six Common Acidic Compounds

2 WVL210 nm

1

2

3

5

6

4

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

4

[min]

Sample

Standard Mixture

Analytical Conditions

Column Develosil XG-C30M, 5 µm

Size 4.6 x 150 mm

Part No. XG30M546150W

Mobile Phase 50 mM Ammonium Formate (pH=2.0)

Flow Rate 1.0 mL/min

Temperature 40°C

Injection Volume 2.0 µL

Detection UV 210 nm

1. Oxalic acid

2. L(+)-Ascorbic acid

3. Maleic acid

4. Citric acid

5. Fumaric acid

6. Uric acid

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Sample

Standard Mixture



Size 4.6 x 150 mm


Mobile PhaseAcetonitrile/0.2% Phosphoric Acid=80/20


Temperature 40°C

Injection Volume 10 µL

Detection UV 210 nm (PDA)

2

4

1 35

6

Fatty Acids



Size 4.6 x 150 mm




Temperature 40°C


Detection UV 210 nm

1. Lauric Acid

2. Linolenic Acid

3. Myristic acid

4. Linoleic acid

5. Oleic Acid

6. Stearic Acid

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Ascorbic Acid

Sample

Standard Mixture



Size 4.6 x 250 mm




Temperature 40°C


Detection CAD

1 23

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00[min]

1. Dehydroascorbic Acid

2. L (+)-Ascorbic Acid

3. Isoascorbic Acid

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Ascorbic and Citric Acid in Drinks

Sample

Drink Brand 1



Size 4.6 x 250 mm




Temperature 40°C


Detection CAD


Column Develosil XG-C1, 5 µm

Size 4.6 x 250 mm

Part No. XGC1546250W

Mobile PhaseAcetonitrile/ 0.2% Phosphoric Acid=2/98


Temperature 30°C



11

2

1. Ascorbic Acid in Drink 1

2. Citric Acid in Drink 1

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Sample

Drink Brand 2



Size 4.6 x 250 mm




Temperature 30°C



Ascorbic and Citric Acid in Drinks (2)

2

1

1. Ascorbic Acid in Drink 2

2. Citric Acid in Drink 2

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SampleSoft Drink

Sample Prep

Filtered soft drink through a 0.45 µm filter



Size 4.6 x 250 mm




Temperature 30°C




Column

1. Develosil HSR AQ C18, 5 µm

2. Develosil HSR C18, 5 µm

3. Develosil HSR C1, 5 µm

Size 4.6 x 150 mm

Part No.

1. 73-546150W

2. 72-546150W

3. 74-546150W

Mobile PhaseA) 10 mM (NH4)2HPO4 (pH=6.0)

B) Acetonitrile

Gradient B) 10-50% (0-10 min)


Temperature 40°C


Detection UV 214 nm

Food Additives in a Soft Drink

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1. Acesulfame K

2. Sodium Benzoate

3. Sodium Saccharin Dihydrate

4. Potassium Sorbate

5. Caffeine

6. Aspartame

Food Additives in a Soft Drink (cont.)

So� Drink

Standard

Column: HSR AQ C18

1 23

45

6

1

5

6

Standard

So� DrinkColumn: HSR C1

23

45

61

1

5

6

Column: HSR C18 So� Drink

Standard1 2

34

5

6

1

5

6

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Sample

Standard Mixture



Size 4.6 x 150 mm



Gradient B% 95 0 (10 min)


Temperature 40°C



Color Additives (1)

11

2

4

3

45 6

78

9

1. Acid yellow 23

2. Acid red 27

3. Indigo carmine

4. Acid yellow 18

5. Sunset yellow

6. Allura red

7. Erythrosine B

8. Phloxine B

9. Rose bengal

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Sample

Standard Mixture



Size 4.6 x 150 mm


Mobile PhaseA) 20 mM ammonium formate

B) Acetonitrile

Gradient

Time A B0 95 1010 0 10020 0 10020.1 95 5


Temperature 40°C


Detection PDA (Max abs.)

Color Additives (2)

3000000 23000000

12

3 4

5

67

2000000

Inte

nsity

[µV]

6

1000000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Retention Time [min]

0

1. Acid yellow 23

2. Acid red 27

3. Acid yellow 18

4. Sunset yellow FCF

5. Allura red

6. Erythrosine B

7. Rose bengal

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Size 4.6 x 150 mm


Mobile PhaseA) 20 mM ammonium formate

B) Acetonitrile

Gradient

Time A B0 95 1010 0 10020 0 10020.1 95 5


Temperature 40°C


Detection PDA (Max abs.)

-10

50

110

0 5 10 15 20 25 30

mV

Time(min)

12 3 4

5

67

8 9

10

Sample

Standard Mixture

1. Tartrazine

2. Amaranth

3. Indigo carmine

4. New coccine

5. Sunset yellow FCF

6. Fast green FCF

7. Brilliant blue FCF

8. Erythrosine B

9. Phloxine B

10. Rose bengal


Column Develosil HSR C18, 5 µm

Size 4.6 x 150 mm

Part No. 72-546150W

Mobile PhaseA) 10 mM (NH4)2HPO4, pH=6.0

B) Acetonitrile

Gradient B) 5%-50% (0-30 min)


Temperature 40°C


Detection UV 254 nm

Color Additives (3)

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Capsaicin

Sample

Capsaicin Standard


Column Develosil Ph-UG, 5 µm

Size 4.6 x 250 mm

Part No. UG15546250W

Mobile Phase Acetonitrile/0.1 % H3PO4=40/60


Temperature 40°C


Detection UV 281 nm

WVL 281WVL:281 nm1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0[min]

1. Capsaicin

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Capsaicin in Pepper Sauce

Sample

Pepper Sauce

Sample Preparation

Dissolve sample in 10 mL of mobile phase (5 mg)

Stir overnight (8+ hours)

Centrifuge (1300 rpm, 10 min)

Filter using a 0.45 µm filter

Injection (20 µL



Size 4.6 x 250 mm




Temperature 40°C


Detection UV 281 nm



Size 4.6 x 250 mm




Temperature 40°C


Detection UV 281 nm

WVL:281 nm

Capsicin

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0[min]

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Capsaicin in Red Peppers

Sample

Red Peppers

Sample Preparation

Dissolve sample in 10 mL of mobile phase (5 mg)

Stir overnight (8+ hours)


Filter using a 0.45 µm filter

Injection (20 µL



Size 4.6 x 250 mm




Temperature 40°C


Detection UV 281 nm

WVL:281 nm

Capsicin

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0[min]

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0 5 10 15 20 25 30 35 40Time(min)

1

2

THF extrac�on

Dichloromethane extrac�on

Acetone extrac�on

Carotenes in Carrots

Sample

Carrots

1. α-carotene

2. ß-carotene

Sample Preparation

Shattered carrots (8.5 g)

Add Solvent (THF, Dichloromethane, or Acetone)


Filter

Injection (5.0 µL)



Size 4.6 x 250 mm




Temperature 40°C


Detection UV 281 nm



Size 4.6 x 250 mm

Part No. 72-546150W

Mobile Phase Methanol/Ethanol=90/10


Temperature 40°C


Detection UV 450 nm

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Catechins in Tea

Sample Preparation

Japanese Green Tea (6.0 g)

Add Water (50 mL)

Leave Overnight (24 hours)

Centrifuge (3800 rpm, 30 minutes)

Dilute with Mobile Phase

(Methanol/0.1% Phosphoric acid=50/50): 10-fold dilution

Filter with a 0.45 µm GHP Membrane

Injection

Sample/Analytes

1. (-) - Epigallocatechin

2. Caffeine

3. (-) - Epigallocatechin gallate

4. (-) - Epicatechin

5. (-) - Epicatechin gallate

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Catechins in Tea (cont.)

Sample

Japanese Green Tea


Column Develosil HSR AQ C18, 5 µm

Size 4.6 x 150 mm

Part No. 73-546150W

Mobile Phase A) 0.1% Phosphoric acid B) Methanol

Gradient

A B Curve

0.0 90 10 530.0 50 50 540.0 50 50 540.1 90 10 550.0 End


Temperature 40°C (Forced air)


Detection UV 280 nm

Mixer Volume 10 µL

1

2

3

4 5

1. (-) - Epigallocatechin

2. Caffeine

3. (-) - Epigallocatechin gallate

4. (-) - Epicatechin

5. (-) - Epicatechin gallate

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1

2

3

4 5

Catechins in Tea (cont.)

Sample

Japanese Green Tea



Size 2.0 x 50 mm

Part No. 73-220050W

Mobile Phase A) 0.1% Phosphoric acid B) Methanol

Gradient

A B Curve

0.0 90 10 54.4 50 50 55.87 50 50 55.88 90 10 57.3 End


Temperature 40°C (Forced air)


Detection UV 280 nm

Mixer Volume 10 µL

1. (-) Epigallocatechin

2. Caffeine

3. (-) Epigallocatechin gallate

4. (-) Epicatechin

5. (-) Epicatechin gallate

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1 2

34 5

Isoflavones

Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 55-546150W

Mobile PhaseA) 0.2% Formic Acid/Water

B) 0.2% Formic Acid/Acetonitrile

Gradient

Time (min) %A %B0.0 100 015.0 20 8015.1 100 0


Temperature 40°C


Detection UV 254 nm

1. Puerarin

2. Daidzin

3. Daidzein

4. Biochanin A

5. Ipriflavone

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Isothiocyanates

Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 72-546150W

Mobile Phase Acetonitrile/0.2% H3PO4=50/50


Temperature 40°C


Detection UV 240 nm

0.0 5.0 10.0 15.0 20.0 25.0 Retention Time [min]

0

100000

200000 In

tens

ity [

µV]

1

2

3

4

5

1. Ethyl Isothiocyanate

2. Allyl Isothiocyanate

3. Propyl Isothiocyanate

4. 2-Phenylethyl Isothiocyanate

5. Phenyl Isothiocyanate

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Maltooligosaccharides

Sample

Standard Mixture


Column Develosil ANIDIUS

Size 4.6 x 250 mm

Part No. ANID546250W

Mobile Phase Acetonitrile/Water=65/35


Temperature 40°C


Detection RI

100000

1

50000

Inte

nsit

y [µ

V] 3

2

45

67

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention Time [min]

0

1. Glucose

2. Maltose

3. Maltotriose

4. Maltotetraose

5. Maltopentaose

6. Maltohexaose

7. Maltoheptaose

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Preservatives (1)

SampleStandard Mixture



Size 4.6 x 150 mm




Temperature 40°C


Detection UV 235 nm

WVL:235 nm4

2

3

2

56

1

3 4

2

3

12

3

5

4

3

4

4

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00[min]

1. p-hydroxybenzoic acid

2. Methyl p-hydroxybenzoate

3. Ethyl p-hydroxybenzoate

4. Propyl p-hydroxybenzoate

5. Butyl p-hydroxybenzoate

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Preservatives (2)

SampleStandard Mixture



Size 4.6 x 150 mm




Temperature 40°C


Detection UV 235 nm



Size 4.6 x 150 mm


Mobile Phase Acetonitrile/0.2% Formic acid=25/75


Temperature 40°C


Detection UV 240 nm

WVL:240 nm1

2

3

4

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0[min]

1. p-hydroxybenzoic acid

2. Benzoic acid

3. Salicylic acid

4. Dehydroacetic acid

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Resveratrol in Red Wine

Sample1. Resveratrol Standard

2. Red Wine

Sample PreparationFiltered red wine through a 0.45 µm filter



Size 4.6 x 150 mm

Part No. 72-546150W



Temperature 40°C

Injection VolumeResveratrol Standard - 5 µL

Red Wine - 10 µL

Detection UV 310 nm

1

min

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Sinigrin

Sample

Standard



Size 4.6 x 150 mm

Part No. 72-546150W



Temperature 40°C

Injection VolumeResveratrol Standard - 5 µL

Red Wine - 10 µL

Detection UV 310 nm


Column/Part No.

Develosil HSR AQ C18, 5 µm

Develosil HSR C1, 5 µm

Develosil C30-UG, 5 µm

Develosil ODS-HG, 5 µm

Develosil ODS, 5 µm

73-546150W

74-546150W

UG17546150W

HG11546150W

ODS0546150W

Size 4.6 x 150 mm

Mobile Phase 10 mM KH2PO4 (pH=2.5)


Temperature 40°C


Detection UV 210 nm

HSR AQ C18, 5um5.884min

4.466min HSR C1, 5um

5.098min C30-UG-5 (5um)

4.032min ODS-HG-5 (5um)

ODS-5 (5um)3.952min

1. Sinigrin

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Saccharides

Sample

Standard Mixture



Size 4.6 x 250 mm




Temperature 40°C


Detection RI

300000

1 2

200000

Inte

nsit

y [µ

V]

3

4

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention Time [min]

0

100000

1. Fructose

2. Glucose

3. Sucrose

4. Maltose

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Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 72-546150W

Mobile Phase Acetonitrile/Methanol=60/40


Temperature 30°C


Detection UV 280 nm

Oil Soluble Vitamins



Size 4.6 x 250 mm




Temperature 40°C


Detection RI

-10

10

30

50

70

90

110

0 5 10 15 20

mV

Time(min)

1

23

4

5

1. Vitamin A acetate

2. Vitamin D3

3. Vitamin E

4. Vitamin E acetate

5. Vitamin K1

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Water-Soluble Vitamins (1)

Sample

Standard Mixture



Size 4.6 x 250 mm


Mobile PhaseA) 0.1% Formic acid + Acetonitrile

B) 0.1% Formic Acid

Gradient B) 25%-60% (15 min) à 60% (30 min)


Temperature 40°C


Detection UV 260 nm

WVL:260 nm

1

2

56

3

4

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0[min]

1. Thiamine

2. Nicotinamide

3. Riboflavin

4. Cyanocobalamin

5. Nicotinic acid

6. Ascorbic acid

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Sample

Standard Mixture



Size 4.6 x 150 mm


Mobile PhaseAcetonitrile/50 mM Ammonium Formate=2/98


Temperature 30°C


Detection UV 260 nm




Size 4.6 x 250 mm


Mobile PhaseA) 0.1% Formic acid + Acetonitrile

B) 0.1% Formic Acid

Gradient B) 25%-60% (15 min) à 60% (30 min)


Temperature 40°C


Detection UV 260 nm

WVL260WVL: nm

1

3

2

4 5 6

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0[min]

1. L(+)-ascorbic acid

2. Orotic acid

3. Nicotinic acid

4. Pyridoxine hydrochloride

5. Nicotinamide

6. Folic acid

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Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 72-546150W

Mobile Phase

A) 20 mM NH4H2PO4, pH=3.0 containing 5 mM CH3(CH2)5SO3Na

B) Methanol containing 5 mM CH3(CH2)5SO3Na

GradientB) 5% (0-5 min), 5%-25% (5-25 min), 25% (25-35 min)


Temperature 40°C


Detection UV 210 nm

-5

20

45

70

0 5 10 15 20 25 30 35

mV

Time (min)

10

8

92

3

4

5

6

7

1

1. L(+)-ascorbic acid

2. Nicotinic acid

3. Nicotinamide

4. Calcium (+)-pantothenate


6. Riboflavin sodium phosphate

7. Folic acid

8. (+)-Biotin

9. Thiamine hydrochloride

10. Cyanocobalamin

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Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 73-546150W

Mobile PhaseA) 25 mM NH4H2PO4 (pH=3.0)

B) Methanol



Temperature 40°C


Detection UV 210 nm




Size 4.6 x 150 mm

Part No. 72-546150W

Mobile Phase

A) 20 mM NH4H2PO4, pH=3.0 containing 5 mM CH3(CH2)5SO3Na

B) Methanol containing 5 mM CH3(CH2)5SO3Na



Temperature 40°C


Detection UV 210 nm

1

2

3 4

5

6

7

8

9

10

1. Thiamin hydrochloride

2. L (+)-Ascorbic acid

3. Nicotinic acid


5. Nicotinamide


7. Cyanocobalamin

8. Folic acid

9. Riboflavin sodium phosphate

10. (+)-Biotin

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Water-Soluble Vitamins via Ion Pair Method

Sample

Standard Mixture

1. Ascorbic acid

2. Nicotinamide

3. Nicotinic acid

4. Thiamine hydrochloride



7. Folic acid

8. Biotin

9. Cyanocobalamin


Column/Part No.1. Develosil XG-C18LC, 5 µm

2. Develosil XG-C18M, 5 µm

55-546150W

XG18M546150W

Size 4.6 x 150 mm

Mobile PhaseA) 0.1% TFA in water

B) 0.1% TFA in methanol

Gradient B) 0-70% (20 min)


Temperature 40°C


Detection UV 210 nm

5

400000

600000

XG-C18LC-51

23

4

5

678 9

200000

400000 In

tens

ity-1

234

5

67

89

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Retention Time [min]

0

XG-C18M-51

24

89

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Sample

Standard Mixture



Size 4.6 x 150 mm

Part No. 72-546150W



Temperature 40°C


Detection UV 325 nm

Vitamin A

min

1

21. Retinoic acid

2. Retinol

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Column1. Develosil XG-C30M, 5 µm

2. Develosil C30-UG, 5 µm

Size 4.6 x 150 mm

Part No.1. XG30M546150W

2. UG17546150W

Mobile Phase Acetonitrile


Temperature 40°C


Detection UV 210 nm

Vitamin D2 and Vitamin D3

Sample

Standard Mixture

WVL:280 nm

1 1

22

1

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

Develosil XG- C3 0 M(5 um)

Develosil C3 0 - UG- 5

[min]

1. Vitamin D2

2. Vitamin D3

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Durable HPLC-RI Analysis of Sugars in Chocolate

Amino columns are a staple in sugar HPLC analysis. However, those amino columns have a critical

issue of low durability. Different amino columns were studied, and their chromatographic properties

were compared in terms of peak shape and number of the theoretical plate number. For durability

evaluation, maltose was used as the sample and the number of theoretical plates was measured

over time. In addition to finding a durable column, a sample preparation method was developed

to avoid hydrolysis effects. An HPLC-RI method was subsequently developed for the identification

and quantification of sugars in chocolate using a durable amino column and the developed sample

preparation method.

Durability Comparison

The following sugar analyses compare the

durability of 5 different NH2 silica-based

columns using maltose. The number of

theoretical plates were measured over time.

The initial value (first run) was set to 100%.

Experimental Conditions:

Five different NH2, 5 µm (4.6 mm ID x 250 mm

long) HPLC columns were used. The mobile

phase consisted of acetonitrile/water (70:30).

The flow rate was 1.0 mL/min and the column

temperature was 40°C. An injection of 5.0 µL

of maltose was used for this experiment. The

HPLC system was connected to a RI detector.

Results:Discussion:

The deterioration was as fast as 120 min for one

column, but the deterioration of the Develosil

NH2 was much slower. After 5,000 minutes,

the theoretical plate number was maintained at

greater than 90% of its initial value.

10

00

80

60

40

20

Mai

nten

ance

Fac

tor

(%)

of T

heor

etic

al

Pla

te N

umbe

r of

Mal

tose

2000 4000

Develosil NH2 (updated)Develosil NH2 (former)

Company A NH2

Company B NH2

Company C NH2

www.develosil.us40

1

2

3

4

5

6

7

0.0 4.0 8.0 12.0 16.0 20.0

Retention Time (minutes)

0

5,000

10,000

Inte

nsity

[µV

]

1

45

6 7

2

3


1

2

3

4

5

6

7

0.0 4.0 8.0 12.0 16.0

0

5,000

10,000

15,000

Inte

nsity

[µV

]

2

3

1

4 56

7

Performance Comparison

The following sugar analyses compared the

performance of 2 different NH2 columns

using maltooligosaccharides. The number of

theoretical places were measured for each

analyte.


Two different NH2, 5 µm (4.6 mm ID x 250 mm

long) HPLC columns were used. The mobile



temperature was 40°C. An injection of 5.0 µL of

a mixture of 7 maltooligosaccharides was used

for this experiment. The HPLC system was

connected to a RI detector.

Results:

Table 1: Comparison of Theoretical Plate Number

Peak NumberDevelosil NH2 NTP

Other Company NH2 NTP

1 9801 5470

2 9160 3655

3 8728 3155

4 8615 2867

5 8214 2351

6 7913 2047

7 8065 2254

Figure 1: HPLC Chromatograph of maltooligosaccharides using a Develosil NH2 column

Figure 2: HPLC Chromatograph of maltooligosaccharides using another Company NH2 column

Discussion:

The Develosil NH2 showed an overwhelming

theoretical plate number in comparison to its

counterpart. The Develosil NH2 theoretical

number remained almost constant, but its

counterpart showed a degradation in plate

number and broader peaks as the elution

continues.

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0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

0

100000

200000

300000

400000

]V

µ[ytis

net

nI

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention Time (minutes)


0

100000

200000

300000

400000

]V

µ[ytis

net

nI

Sucrose

Fructose

Glucose

Sucrose

ACN Treatment

TCA Treatment

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

0

100000

200000

300000

400000

]V

µ[ytis

net

nI

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention Time (minutes)


0

100000

200000

300000

400000

]V

µ[ytis

net

nI

Sucrose

Fructose

Glucose

Sucrose

ACN Treatment

TCA Treatment

Analysis of Sugars in Chocolate

Sample Preparation:In a 50 mL centrifuge tube, add 20 mL of water

to 1 mL of chocolate, and then add 20 mL of

hexane. Centrifuge for 15 minutes at 3900 rpm.

Transfer the aqueous phase from the mixture into

another 50 mL centrifuge tube. Add a subsequent

20 mL of hexane to the aqueous mixture and stir.

Centrifuge for 15 minutes at 3900 rpm. Add and

stir ACN into mixture. The amount of ACN to be

added in a 1:1 (v:v) ratio with water. Centrifuge

for 5 minutes at 13200 rpm. Take supernatant as

sample. Push sample through 0.45 µm filter.

Why is ACN used instead of TCA as the deproteinizing solvent?The figures below represent chromatograms that

demonstrate the effects of using trichloroacetic

acid (TCA, Fig. 3) and acetonitrile (ACN Fig 4) as

the deproteinizing solvent. Analytical conditions

are the same as the analysis of sugars in

chocolate using fructose, sucrose, and glucose

as standards.

The use of TCA for deproteinization step of

chocolate sample preparation show that the peak

of sucrose decreased overtime while the peaks

of fructose and glucose increased overtime. This

is the result of the hydrolysis of TCA. The use

of ACN did not demonstrate this hydrolysis and

presented a single peak.

Table 2: Analytical Conditions

Column Develosil NH2, 5 µm

Size 4.6 x 250 mm



Temperature 40°C

Sample

1. Fructose

2. Glucose

3. Sucrose


Detector RI

Figure 3: HPLC Chromatograph using TCA as deproteinizing solvent

Figure 4: HPLC Chromatograph using ACN as deproteinizing solvent

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Analysis of Sugars in Chocolate


A Develosil NH2, 5 µm (4.6 mm ID x 250 mm

long) HPLC columns was used. The mobile



temperature was 40°C. An injection of 5.0 µL of

the prepared sample was used for this analysis.

The HPLC system was connected to a RI

detector.

Results:

Glucose and fructose were detected in the

chocolate sample. A calibration curve was

prepared by the addition recovery test method.

The concentration vs absorbance plot was

calculated and the concentration of each

saccharide in the chocolate sample using the

calibration curve.

Conclusion:

As a result of the analysis of sugars in chocolate,

only sucrose was identified. As a result, it was

possible to obtain a good linearity. The correlation

coefficient (R2) resulted in values 0.9996 and

0.9998 for glucose and sucrose respectively.

In addition, the recovery rate was 100% for

sucrose. For chocolate samples, pretreatment

is important and complicated. Analytical errors

tend to occur due to the complexities but using

the Develosil NH2 and an RI detector produced

quality, quantitative results.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Retention Time (minute)

0

100000

200000

]V

µ[ytis

net

nI

SucroseUnidentified peak

Figure 5. Typical Chromatogram of Chocolate Samples

Figure 6. Calibration Curve of Sucrose and Spiked Chocolate SampleYellow line is sucrose level in chocolate and spiked.Blue line shows calibration curve of sucrose standard.

y = 71061x - 3390.8R² = 0.9993

y = 72079x + 628456R² = 1

-100000

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 1 2 3 4 5

(mg/mL)

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Product Information

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ODS Functional Group

HSR C18

Features: Your go-to column for all HPLC analyses. With high particle surface area, it offers high selectivity and high separation performance. The Develosil HSR C18 is subjected to a special end cap treatment that offers good peak shape in acid, basic or neutral conditions.

Bonding and End Capping: Polymeric ODS with special TMS end-capping

HSR AQ C18

Features: An ODS column that allows you to separate your high polarity compounds in reverse phase operations. The Develosil HSR AQ C18 gives you gives you maximum performance even in 100% aqueous conditions.

Bonding and End Capping: Monomeric/Polymeric ODS with special TMS end-capping

XG-C18M

Features: An ODS column designed that offers low column back pressure and fast equilibrium times. With a specialized hydrophilic polar group end capping, the Develosil XG-C18M offers a greater retention for compounds with higher polarity.

Bonding and End Capping: Monomeric ODS with polarized end-capping

XG-C18LC

Features: Having a controlled lower carbon content and a specialized hydrophilic polar group end capping, the Develosil XG-C18LC can separate high polarity compounds.

Bonding and End-Capping: Polymeric: ODS with polarized end-capping

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C30 Functional Group

C30-UG/ RPAQUEOUS

Features: Our most well-known column! The Develosil C30-UG, also known as the RPAQUEOUS, works with 100% aqueous mobile phase and gives added retention highly polar compounds.

Bonding and End-Capping: Monofunctional Triacontyl with double TMS end-capping

XG-C30M

Features: A C30 column designed for fast equilibration time and low column backpressure. The Develosil XG-C30M is subjected to specialized polarized end for added retention for highly polar compounds.

Bonding and End-Capping: Monofunctional Triacontyl with specialized polarity end-capping

Specialty Functional Group

HSR C1

Features: A C1 column that allows you to separate your high polarity compounds in reverse phase operations. The Develosil HSR C1 offers faster elution for hydrophobic compounds.

Bonding and End-Capping: Methyl radical with special TMS end-capping

XG-C1Features: With specialized hydrophilic polar end-capping and high surface area, the C1 gives you maximum retention and separation.

Bonding and End-Capping: Methyl radical with polarized end-capping

Ph-UG

Features: The Develosil Ph-UG added extra π electron interactions to increase aromatic selectivity. With double-end capping, it limits problems with conventional residual silanol groups.

Bonding and End-Capping: Phenyl radical with double TMS end-capping

ANIDIUS

Features: Column specifically designed for HILIC operations. Diol and amine phases are bonded onto the silica base with proprietary technology to maximize separations for high polarity compounds.

Bonding and End-Capping: Proprietary bonding and end-capping technology

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Column Selection for your Method Development

To start your method development, simply follow one of the paths for your analyte and find your recommended column:

Reverse Phase Method Development

Normal Phase Method Development

SEC Method Development

Small Organic Molecules



Standard Analysis: Develosil HSR C18 Develosil HSR AQ C18

Silica Phase Chemistry: Develosil Silica 30 Develosil Silica 60 Develosil Silica 100

Develosil 100 Diol Develosil 300 Diol

Develosil 300 Diol

Cyano Phase Chemistry: Develosil NH2

Diol Phase Chemistry: Develosil 100 Diol

Develosil HSR C18 Peptide Develosil 300 ODS-HG Develosil 300 C8-HG Develosil 300 C4-HG

Accelerated Elusion: Develosil HSR C8 Develosil HSR C1

Large Organic Molecules



These columns are recommended as the starting column based off your analyte chemistry Develosil has a wide selection of columns to help you improve your retention and selectivity. Please contact us for more information about our full product lineup.

[email protected] | (858) 800-2433 47

Column Selection for your Application

Have a certain application in mind? See below for our recommended column based on your application.

Organic Acid Applications

Carbohydrates Applications

Vitamin Applications

Reverse Phase

Reverse Phase HILIC

Water Soluble Vitamins

Develosil HSR AQ C18 Develosil HSR C1 Develosil RPAQUEOUS-AR

Develosil HSR AQ C18 Develosil RPAQUEOUS

Develosil NH2Develosil ANIDIUS

Reverse Phase: Develosil HSR AQ C18 Develosil RPAQUEOUS Develosil RPAQUEOUS-AR

Normal Phase and HILIC: Develosil 30 Develosil 60 Develosil 100 Develosil NH2 Develosil ANIDIUS

HILIC: Develosil NH2 Develosil ANIDIUS

Fat-Soluble Vitamins

These columns are recommended as the starting column based off your application. Develosil has a wide selection of columns to help you improve your retention and selectivity. Please contact us for more information about our full product lineup and visit our website for application notes.

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About DevelosilNomura Chemical has been producing Develosil HPLC columns since 1979. Nomura Chemical

manufactures the entire Develosil series from the silica gel packing material to the final packed column

in order to provide customers with high reproducibility and quality separations.

In November 2017, Develosil USA was established. Develosil USA was established to provide local

support for North American customers of all HPLC columns manufactured by Nomura Chemical

Co., Ltd as well as gather customer feedback on the Develosil product line. Develosil USA will be

working towards obtaining analysis data from leading edge applications to incorporate them into

future Develosil product lines.

Develosil HPLC columns have been a vital tool for the food industry. They have been used in research

that has been published in journals such as Food Chemistry and Journal of Functional Foods. Our

food application portfolio grows each day. We continue to support industry needs by developing new

products and offering application and method development services.

Nomura Chemical Headquarters in Japan

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