food application notes - develosil usa · 8. erythrosine b 9. phloxine b 10. rose bengal analytical...
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Food Application Notes
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CONTRACT ANALYSISFREE* Column Transfer Service
*Restrictions
Detectors available for HPLC analysis is UV detector, fluorescence detector, and differential refractometer.
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Development Service.
Develosil USA is offering a column transfer service with FREE* analytical testing to help you find the best Develosil column for your HPLC and UHPLC processes.
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To get started, please contact us at info@develosil.us or visit www.develosil.us
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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
Analytical Conditions
Column Develosil XG-C18M, 5 µm
Size 4.6 x 150 mm
Part No. XG18M546150W
Mobile PhaseAcetonitrile/0.2% Phosphoric Acid=80/20
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 10 µL
Detection UV 210 nm (PDA)
2
4
1 35
6
Fatty Acids
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. 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
Analytical Conditions
Column Develosil XG-C30M, 5 µm
Size 4.6 x 250 mm
Part No. XG30M546250W
Mobile Phase 20 mM Ammonium Formate (pH=2.0)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil XG-C30M, 5 µm
Size 4.6 x 250 mm
Part No. XG30M546250W
Mobile Phase 20 mM Ammonium Formate (pH=2.0)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
Detection CAD
Analytical Conditions
Column Develosil XG-C1, 5 µm
Size 4.6 x 250 mm
Part No. XGC1546250W
Mobile PhaseAcetonitrile/ 0.2% Phosphoric Acid=2/98
Flow Rate 1.0 mL/min
Temperature 30°C
Injection Volume 10.0 µL
Detection UV 210 nm (PDA)
11
2
1. Ascorbic Acid in Drink 1
2. Citric Acid in Drink 1
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Sample
Drink Brand 2
Analytical Conditions
Column Develosil XG-C1, 5 µm
Size 4.6 x 250 mm
Part No. XGC1546250W
Mobile PhaseAcetonitrile/ 0.2% Phosphoric Acid=2/98
Flow Rate 1.0 mL/min
Temperature 30°C
Injection Volume 10.0 µL
Detection UV 210 nm (PDA)
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
Analytical Conditions
Column Develosil XG-C1, 5 µm
Size 4.6 x 250 mm
Part No. XGC1546250W
Mobile PhaseAcetonitrile/ 0.2% Phosphoric Acid=2/98
Flow Rate 1.0 mL/min
Temperature 30°C
Injection Volume 10.0 µL
Detection UV 210 nm (PDA)
Analytical Conditions
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 1.0 µL
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
Analytical Conditions
Column Develosil XG-C30M, 5 µm
Size 4.6 x 150 mm
Part No. XGC1546250W
Mobile PhaseAcetonitrile/ 0.2% Phosphoric Acid=2/98
Gradient B% 95 0 (10 min)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
Detection UV 254 nm (PDA)
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
Analytical Conditions
Column Develosil XG-C1, 5 µm
Size 4.6 x 150 mm
Part No. XGC1546250W
Mobile PhaseA) 20 mM ammonium formate
B) Acetonitrile
Gradient
Time A B0 95 1010 0 10020 0 10020.1 95 5
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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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Analytical Conditions
Column Develosil XG-C1, 5 µm
Size 4.6 x 150 mm
Part No. XGC1546250W
Mobile PhaseA) 20 mM ammonium formate
B) Acetonitrile
Gradient
Time A B0 95 1010 0 10020 0 10020.1 95 5
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 5.0 µL
Detection UV 254 nm
Color Additives (3)
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Capsaicin
Sample
Capsaicin Standard
Analytical Conditions
Column Develosil Ph-UG, 5 µm
Size 4.6 x 250 mm
Part No. UG15546250W
Mobile Phase Acetonitrile/0.1 % H3PO4=40/60
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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
Analytical Conditions
Column Develosil Ph-UG, 5 µm
Size 4.6 x 250 mm
Part No. UG15546250W
Mobile Phase Acetonitrile/0.1 % H3PO4=40/60
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
Detection UV 281 nm
Analytical Conditions
Column Develosil Ph-UG, 5 µm
Size 4.6 x 250 mm
Part No. UG15546250W
Mobile Phase Acetonitrile/0.1 % H3PO4=40/60
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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)
Centrifuge (1300 rpm, 10 min)
Filter using a 0.45 µm filter
Injection (20 µL
Analytical Conditions
Column Develosil Ph-UG, 5 µm
Size 4.6 x 250 mm
Part No. UG15546250W
Mobile Phase Acetonitrile/0.1 % H3PO4=40/60
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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)
Centrifuge (3800 rpm, 20 min)
Filter
Injection (5.0 µL)
Analytical Conditions
Column Develosil Ph-UG, 5 µm
Size 4.6 x 250 mm
Part No. UG15546250W
Mobile Phase Acetonitrile/0.1 % H3PO4=40/60
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
Detection UV 281 nm
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 250 mm
Part No. 72-546150W
Mobile Phase Methanol/Ethanol=90/10
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 5.0 µL
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
Analytical Conditions
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
Flow Rate 1.0 mL/min
Temperature 40°C (Forced air)
Injection Volume 10 µL
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
Analytical Conditions
Column Develosil HSR AQ C18, 2 µm
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
Flow Rate 0.474 mL/min
Temperature 40°C (Forced air)
Injection Volume 0.8 µL
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
Analytical Conditions
Column Develosil HSR C18, 5 µm
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
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 10 µL
Detection UV 254 nm
1. Puerarin
2. Daidzin
3. Daidzein
4. Biochanin A
5. Ipriflavone
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Isothiocyanates
Sample
Standard Mixture
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 150 mm
Part No. 72-546150W
Mobile Phase Acetonitrile/0.2% H3PO4=50/50
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil ANIDIUS
Size 4.6 x 250 mm
Part No. ANID546250W
Mobile Phase Acetonitrile/Water=65/35
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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
Analytical Conditions
Column Develosil XG-C18M, 5 µm
Size 4.6 x 150 mm
Part No. XG18M546150W
Mobile Phase Acetonitrile/Water=50/50
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil XG-C18M, 5 µm
Size 4.6 x 150 mm
Part No. XG18M546150W
Mobile Phase Acetonitrile/Water=50/50
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
Detection UV 235 nm
Analytical Conditions
Column Develosil XG-C18M, 5 µm
Size 4.6 x 150 mm
Part No. XG18M546150W
Mobile Phase Acetonitrile/0.2% Formic acid=25/75
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 150 mm
Part No. 72-546150W
Mobile Phase Acetonitrile/Water=20/80
Flow Rate 1.0 mL/min
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
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 150 mm
Part No. 72-546150W
Mobile Phase Acetonitrile/Water=20/80
Flow Rate 1.0 mL/min
Temperature 40°C
Injection VolumeResveratrol Standard - 5 µL
Red Wine - 10 µL
Detection UV 310 nm
Analytical Conditions
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil ANIDIUS
Size 4.6 x 250 mm
Part No. ANID546250W
Mobile Phase Acetonitrile/Water=80/20
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 150 mm
Part No. 72-546150W
Mobile Phase Acetonitrile/Methanol=60/40
Flow Rate 1.0 mL/min
Temperature 30°C
Injection Volume 1.0 µL
Detection UV 280 nm
Oil Soluble Vitamins
Analytical Conditions
Column Develosil ANIDIUS
Size 4.6 x 250 mm
Part No. ANID546250W
Mobile Phase Acetonitrile/Water=80/20
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 20 µL
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
Analytical Conditions
Column Develosil ANIDIUS
Size 4.6 x 250 mm
Part No. ANID546250W
Mobile PhaseA) 0.1% Formic acid + Acetonitrile
B) 0.1% Formic Acid
Gradient B) 25%-60% (15 min) à 60% (30 min)
Flow Rate 0.5 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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
Analytical Conditions
Column Develosil XG-C18M, 5 µm
Size 4.6 x 150 mm
Part No. XG18M546150W
Mobile PhaseAcetonitrile/50 mM Ammonium Formate=2/98
Flow Rate 1.0 mL/min
Temperature 30°C
Injection Volume 2.0 µL
Detection UV 260 nm
Water-Soluble Vitamins (2)
Analytical Conditions
Column Develosil ANIDIUS
Size 4.6 x 250 mm
Part No. ANID546250W
Mobile PhaseA) 0.1% Formic acid + Acetonitrile
B) 0.1% Formic Acid
Gradient B) 25%-60% (15 min) à 60% (30 min)
Flow Rate 0.5 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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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Water-Soluble Vitamins (3)
Sample
Standard Mixture
Analytical Conditions
Column Develosil HSR C18, 5 µm
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 5 µL
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
5. Pyridoxine hydrochloride
6. Riboflavin sodium phosphate
7. Folic acid
8. (+)-Biotin
9. Thiamine hydrochloride
10. Cyanocobalamin
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Sample
Standard Mixture
Analytical Conditions
Column Develosil HSR AQ C18, 5 µm
Size 4.6 x 150 mm
Part No. 73-546150W
Mobile PhaseA) 25 mM NH4H2PO4 (pH=3.0)
B) Methanol
GradientB) 5% (0-2 min), 5%-50% (2-7 min), 50% (7-10 min)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 1.0 µL
Detection UV 210 nm
Water-Soluble Vitamins (4)
Analytical Conditions
Column Develosil HSR C18, 5 µm
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 5 µL
Detection UV 210 nm
1
2
3 4
5
6
7
8
9
10
1. Thiamin hydrochloride
2. L (+)-Ascorbic acid
3. Nicotinic acid
4. Pyridoxine hydrochloride
5. Nicotinamide
6. Calcium (+)-pantothenate
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
5. Pyridoxine hydrochloride
6. Calcium (+)-pantothenate
7. Folic acid
8. Biotin
9. Cyanocobalamin
Analytical Conditions
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)
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 5 µL
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
Analytical Conditions
Column Develosil HSR C18, 5 µm
Size 4.6 x 150 mm
Part No. 72-546150W
Mobile Phase Acetonitrile/Water=95/5
Flow Rate 0.5 mL/min
Temperature 40°C
Injection Volume 2.0 µL
Detection UV 325 nm
Vitamin A
min
1
21. Retinoic acid
2. Retinol
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Analytical Conditions
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
Flow Rate 1.0 mL/min
Temperature 40°C
Injection Volume 2.0 µL
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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info@develosil.us | (858) 800-2433 39
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
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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
Retention Time (minutes)
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.
Experimental Conditions:
Two different NH2, 5 µm (4.6 mm ID x 250 mm
long) HPLC columns were used. The mobile
phase consisted of acetonitrile/water (60:40).
The flow rate was 1.0 mL/min and the column
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)
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)
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
Mobile Phase Acetonitrile/Water=70/30
Flow Rate 1.0 mL/min
Temperature 40°C
Sample
1. Fructose
2. Glucose
3. Sucrose
Injection Volume 5.0 µL
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
Experimental Conditions:
A Develosil NH2, 5 µm (4.6 mm ID x 250 mm
long) HPLC columns was 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
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
Small Organic Molecules
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
Large Organic Molecules
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.
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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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