Nutrition Study and Quality

Assurance Analysis by
HPLC
Yee Ghee, Lim
Chromatography Product Line Leader
Korea, Oceania and South East Asia

2009 PerkinElmer

Why HPLC for Food Analysis?

Precise and quantitative ( Peak area RSD < 1%)
Analyzes multi-components in complex matrices
Amenable to thermally labile and nonvolatile components
Rapid analysis (5 - 30 minutes)
Sensitive and selective detection (pg - ng levels)
Flexible - wide selection of column, mobile phase, and detectors
Highly automated

Present Role of HPLC

in Nutrition Labeling/Study
Mandatory Nutrients
Sugars (total)
Vitamins (A and C)
Cholesterol

Voluntary Nutrients
Vitamins (D, E, K, B6, B12, thiamine, riboflavin, niacin, folate, biotin, pantothenic acid)
Sugar alcohols
Fats

In the next section, we will discuss HPLC analysis of sugars, vitamins, cholesterol and
fats.

HPLC Analysis of Carbohydrates

Background

Common carbohydrates are

Monosaccharides (glucose, fructose)
Disaccharides (sucrose, maltose, lactose)
Trisaccharides (raffinose) and polysaccharides (starch)

HPLC offers a direct, quantitative method for carbohydrates

Requires specialty cationic resin-based columns with column oven and refractive index
detection
UV detection at low wavelengths (195 nm) can be used but is prone to interferences
Can also use amino column with acetonitrile/water (80/20) though sensitivity is lower (AOAC
Method)
Reference: F. Scott, HPLC determination of carbohydrates in foods, in Food Analysis by HPLC, L. Nollet (ed.),
Marcel Dekker, New York, 1992.

HPLC of Carbohydrates

Using fixed-ion resin-based columns

Different resins (Ca++, Pb++, H+, Ag+, etc.) are used for different sample types
Calcium is the most common form ; water used as the mobile phase at 85 C, (e.g.,
BioRad Aminex HPX-87C, or PE Brownlee Polypore CA)

Mixed-mode separation which includes

Size-exclusion, ion-exclusion, ligand exchange, and hydrophobic interaction (solutes
interact with the polystyrene backbone)

Excellent performance and reliability with RI detection

Precision (peak area 0.7%, retention time 0.1% RSD)
Sensitivity (10-20 ng), linearity (0.050-800 g)

HPLC Analysis of Sugars in Wine

Using Amino Column

Standards

Red Wine
1

Column:

Mobile Phase: CH3CN/H2O (78:22)

3 4

PE Brownlee
Spheri-5 Amino
(100 X 4.6 mm i.d.)

Flow Rate:

2.0 mL/min

Detection:

RI

1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Lactose

Analysis of Sugars in Soft Drinks

Using Ca++ resin-based column

Vitamin Analysis by HPLC

Vital substances for healthy growth of many organisms
Water-soluble vitamins (WSV) -- Ascorbic acid (Vit. C), Niacin, Niacinamide(Vit. B2),
Pyridoxine (Vit. B6), Thiamine (Vit B1), Folic acid, and Riboflavin, pantothenic acid,
cyanocobalamin (Vit. B12) and Biotin
Fat-soluble vitamins (FSV) --Vitamins A, D2, D3, E, and K

HPLC - best quantitative method for multivitamins

Requires reduced-activity C8 and C18 columns with UV detection
Ion-pair reagent used in the mobile phase for WSV separation

Reference: W. E. Lambert et al. Quantitative determination of WSVs using HPLC, in Food

Analysis by HPLC, L. Nollet (ed.), Marcel Dekker, New York, 1992.

Perkin-Elmer HPLC System

for Vitamin Analysis

Series 200 binary pump

Series 200 autosampler or manual injector
PE Brownlee reduced-activity C8 and C18 3-m columns with guard column
Series 200 Column oven
200 UV/Vis or diode array UV detector
LC Control and data handling
TotalChrom and Iris Software

Sample Extraction Procedure

For WSVs in tablets or feed premix

10

1.

Grind one multivitamin tablet or 1 g of premix with a mortar and pestle. Then transfer the contents into a
125-mL Erlenmeyer flask

2.

Pour exactly 10 mL of 1% ammonia in DMSO into the ground powder and sonicate it in an ultrasonic bath
(>200 Watts) at 40 C for two minutes

3.

Add exactly 90 mL of 2% acetic acid in water to the mixture. Stir it with a magnetic stir bar for one minute

4.

Filter the extract while warm through a 0.45-m membrane filter into amber vials. Analyze it immediately
by LC

5.

LC Conditions:
PE reduced-activity 3-m C8 column (83 mm x 4.6 mm i.d.)
15% methanol, 85% [10 mM hexanesulfonate (Sigma), 1% acetic acid, 0.13% triethylamine in water], 1.5 mL/min at
35 C
UV absorbance at 275 nm

Analysis of WSVs in Animal Feed Premix

11

Analysis of FSVs in Animal Feed Premix

12

Summary of Validation Data

For multivitamin analysis

Performance Parameter
Precision
Retention Time
Peak Area
Assay (tablet)
Accuracy
(Recovery of Spikes)
Limit of Detection
(S/N = 3)
Selectivity

WSV

FSV

< 0.3%
< 1.0%
< 3% (10% for Vit. C)
90 - 100%

<0.15%
< 1.1%
< 3%
98% Vit. A
87% Vit. E
< 1 ng (Vit. A and E)
< 10 ng (Vit. E)
PI < 1.1, R > 5
n > 8 000

0.6 - 1.2 ng
PI < 1.1, R > 3
n > 6 000

Range

1 - > 1000 ng

Linearity
Ruggedness Towards
Mobile Phase Preparation
Column Lifetime

r > 0.9995

1 - > 10 000 ng (Vit. A)

5 - > 20 000 ng (Vit. E)
r > 0.999

5%
> 1 000 injections

3%
> 1 000 injections

PI = Purity Index, r = Coeff. of Linear Regression, and n = Column Plate Count.

* Except for Vitamin C, which is unstable in a water solution.

13

Lipids

Background
Fats and oils are made up of triesters of glycerol with fatty acids (triglycerides)
Fatty acids are commonly analyzed after sample transesterification by GC as their
methyl esters; however, free or derivatized fatty acids can also be analyzed by
HPLC
Phospholipids (lecithin, cephalin or phosphotidyl-inositol) from soybeans are used
as food additives
Common sterols are cholesterols and phytosterols (sitosterols and stigmasterol)
Reference: D. Marini, HPLC of Lipids in Food Analysis by HPLC, L. Nollet (ed.), Marcel Dekker, New York,
1992.

14

HPLC Analysis of Triglycerides

HPLC analysis of triglycerides is simple and quantitative
Edible oils can be injected directly as a 5% solution in acetone
Non-aqueous reversed-phase (NARP) mode is used with a C18 column and a mobile
phase of acetone/acetonitrile (70/30)

Refractive Index (RI) Detection is commonly used

Alternately, several detectors can be used with gradient elution
Infrared(C-H stretch 3.4 and C=O stretch at 5.7 )
Mass evaporative detector (nubilization light-scattering)(ELSD)
UV detection (220 nm) is subject to interferences
Note: High-temperature capillary GC (metal coated) with a programmable-temperature vaporization (PTV)
injector was found useful for this application
Reference: M. W. Dong et al., Improved separation of natural oil triglycerides using columns packed with 3m particles, J. Amer. Oil Chem. Soc., 60 (1983) 788.

15

Determination of Cholesterol

Phytosterols and Tocopherols

Column:

Silica
(5-m, 150 x 4.6 mm)
Mobile Phase: Methanol
Flow Rate:
2.5 mL/min
Detection:
UV at 210 nm
Sample:
Mixture of standards

1. -tocopherol
2. -tocopherol
3. -tocopherol
4. Cholesterol
5. Stigmasterol
6. Campesterol
7. -sitosterol

Minutes
Chromatogram from: H. E. Indyk, Simultaneous LC determination of cholesterol, phytosterols
and tocopherols in foods, Analyst, 115 (1990)1525.
16

Triglycerides in Palm Olein (Oil)

by NARP and RI detection

POO

POP

Column:

Two Pecosphere-3 C18

(3-m,83 x 4.6 mm)

Mobile Phase: Acetone/acetonitrile (7:3)

PPL
PLO

LOO

OOO
SPO
SOO

Flow Rate:

3.5 mL/min

Detection:

RI

Sample:

20 L of 5% Palm Olein in
Acetone

Key
OOO = Triolean
L = Linoleic Acid
P = Palmitic Acid
S = Stearic Acid

Minutes
Reference: M. W. Dong etal., Improved separation of natural oil triglycerides using columns packed with 3-m
particles, J. Amer. Oil Chem. Soc., 60 (1983) 788.
17

Tocopherols (Vitamin E)

in Palm Olein

Column:

Pecosphere 5 x 15C Silica

(150 x 4.6 mm)

Mobile Phase: 0.74% Ethanol in Hexane

Flow Rate:

2.0 mL/min

Detection:

Fluorescence at 298 nm
(Ex) / 325 nm (Em)

Sample:

5 L of 0.1% of Crude Palm

Olein in Hexane

1. -tocopherol
2. -tocotrienol
3. -tocotrienol
4. -tocotrienol
PE Brownlee LC Applications Notes LCFD-15.
18

Food Applications

by HPLC

Natural food components

Carbohydrates
Lipids, triglycerides and cholesterols
Fatty acids and organic acids
Proteins, peptides and amino acids

Mode

Detection

IEC, RP
RP, LSC
IEC, RP
RP, IEC

RI
UV, RI
UV, RI
UV/Vis, FL

RP, LSC

UV
RP
UV/Vis
RP

Food additives
Acidulants, sweeteners, flavors, emulsifiers
Anitoxidants and preservatives
Colors and dyes
Vitamins

RP

Contaminants
Mycotoxins (aflatoxins)
Pesticide and drug residues
PAHs and nitrosamines

19

RP
RP
RP

FL, UV
UV, FL
UV, FL

UV
UV, FL

HPLC Analysis

Of fatty and organic acids

Free fatty and organic acids are analyzed by
C18 column with acidified mobile phase, or
Hydrogen ion resin-based column with dilute H2SO4 as mobile phase
Detection by UV at 210 nm or RI

Pre-column derivatization can improve the chromatography and detection sensitivity of

fatty acids
Gradient analysis can be used to separate these fatty acid derivatives
Bromophenacyl esters (UV)
2-nitrophenyl hydrazides (UV)
anthrylmethylesters (fluorescence)

20

HPLC of Un-Derivatized

Long-chain fatty acids with UV detection

18:3 (cis)
16:1 (cis)

Column:
16:1
(trans)

18:2 (cis)
18:2
(trans)
18:0
18:1 (cis)
18:1
(trans) 18:0

C18 (5 m, 250 x 4.6 mm)

Mobile Phase: THF/ACN/0.1% H3PO4

(22:50:28)
Flow Rate:

1.5 mL/min at 35 C

Detection:

220 nm

Sample:

Mixture of fatty acid

standards

D. Marini, HPLC of Lipids, in Food Analysis by HPLC, L. Nollet (ed.), Marcel Dekker, NY, 1992.
21

p-Bromophenacyl Esters

of Fatty Acids with UV Detection

Column:

Pecosphere- 3 C18
(3 m, 100 x 4.6 mm i.d.)

Mobile Phase: 40% Acetonitrile/H2O

(pH 2.5) to 100%
Acetonitrile in 10 min;
Convex Gradient
Flow Rate:

2.5 mL/min

Detection:

UV at 254 nm

Sample:

20-200 ng fatty acid each

Peak numbers designate the number of

carbons in the fatty acid chain

Minutes
PE Brownlee Applications Note LCFD-4.
22

Organic Acids

Common organic acids:

Unsubstituted - formic, acetic, propionic, butyric
Substituted - glycolic, lactic, pyruvic, glyoxylic
Di- or Tricarboxylic - oxalic, succinic, fumaric, maleic, malic, & citric acid

Occurrence, use and analysis

Occur naturally in foods as a result of biochemical metabolic process, hydrolysis or bacterial
growth
Also added as stabilizers or preservatives and to endow flavor, taste or aroma (e.g., propionic acid,
citric acid)
Some organic acids are indicators for ripeness, bacterial activity or spoilage
Analyzed by HPLC with resin-based columns with RI or UV @ 210 nm
D. Blanco Gomis, HPLC of organic acids, in Food Analysis by HPLC, L. Nollet (ed.), Marcel Dekker, New York, 1992.

23

Organic Acid Analysis

Using a resin-based column

1
4

Column:

Polypore H
(10-m, 220 x 4.6 mm)

Detection:

UV at 210 nm

Mobile Phase: 0.01 N H2SO4

Flow Rate:

0.15 mL/min at room

temp.
Peak Identification:

24

20
Time (min)

40

1. Formic acid
2. Acetic acid
3. Propionic acid
4. Benzoic acid

Chromatogram courtesy of Dr. Daniel Tan of PKI Singapore

Organic Acids, Sugars and Ethanol

ethanol

acetic

lactic

shikimic

malic

ethanol

lactic

acetic

ethanol

30

Detection: RI/UV at 210nm

Sample: 10 L of sample
diluted 1:4 with
mobile phase

ethanol

acetic

Column: Two Polypore H

(220 x 4.6 mm i.d.)
Flow Rate: 0.2 mL/min

UV
shikimic

tartaric
citric

fructose
malic

acetic

30

Mobile Phase: 0.01N H2SO4

20
Time (min)

lactic
lactic

succinic

citric
glucose
tartaric
fructose
malic

oxalic

25

20
Time (min)

UV
20
Time (min)

glycerol

citric

10

Standards

10

citric

succinic
glycerol

malic

10

fructose

citric

RI Intensity

Grape
must

glucose

White
wine

tartaric

tartaric

ethanol

in grape musts and wines

30

PE Brownlee LC Applications Notes

LCFD-27.

Proteins and Peptides

General Information

Total protein in food is commonly estimated by

Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis

HPLC can furnish protein profile and speciation information

HPLC can be used to further characterize specific proteins via peptide mapping
and amino acid sequencing techniques
Protein analysis is important for these food products
Meat, fish, dairy, cereal, fruit and vegetables

HPLC modes used include IEC, SEC, RPC and affinity with UV detection at 215
nm

J. Vervaeck and A. Huyghebaert, HPLC of Food Proteins, in Food Analysis by HPLC,

L. Nollet (ed.), Marcel Dekker, New York, 1992.

26

Classification of Food Proteins

Cereal proteins

Albumins (extractable in water)

Globulins (extractable in dilute salt solution)
Prolamins (extractable in aqueous solution of alcohol, gliadin in wheat and zein in corn)
Analysis of cereal proteins is important for:
Amount and type of proteins that influence functional properties (e.g., baking quality)
For development of improved grain genotypes

Milk proteins
Caseins - small MW colloidal proteins precipitated at pH 4-6
Whey proteins soluble in milk serum phase (by-products of the cheese making process)

27

Proteins in Egg Whites

by RPLC

Column:

Pecosphere HCODS C18

HS-5 (5 m x 4.6 mm i.d.)

Mobile Phase: A: Acetonitrile (ACN)

B: Aqueous Solution of
0.1% Trifluoroacetic acid (TFA)
Linear Gradient of 20 to
70% A in 40 min
Flow Rate:

1 mL/min

Detection:

UV at 220 nm

Sample:

20 L of 10% Soln Egg White in

Water

PE Brownlee LC Applications Notes LCFD-13.

28

Separation of a Complex Peptide Mixture

using a C18 narrowbore column
Tryptic Map of Glycoprotein Human IgA

29

Column:

Aquapore RP-300 ODS

(7-m, 300 A, 220 x 2.1 mm)

Mobile Phase:

A: 0.1% TFA in ACN

B: 0.1% TFA
2 - 70% A in 45 min

Flow Rate:

0.25 mL/min

Amino Acids
Amino acid analysis is useful for assessment of the nutritional value of foods and
feedstuffs
Essential amino acids are
- methionine
- threonine
- leucine
- tryptophan

- cystine
- valine
- phenylalanine

- lysine
- isoleucine
- tyrosine

Glutamic acid is a commercial flavor enhancer

Two types of amino acid analysis by HPLC
Post-column derivatization - IEC free amino acids followed by ninhydrin or OPA reactions to
form derivatives
Precolumn derivatization - RPLC analysis of OPA, FMOC, PITC, Dansyl or dabsyl
derivatives of amino acids
Reference: J. White, et al. HPLC analysis of amino acids, in Food Analysis by HPLC, L. Nollet (ed.), Marcel
Dekker, New York, 1992.

30

Configuration of an HPLC System

As a dedicated amino acid analyzer

Reference: Post-column LC system for amino acid analysis,

B-AA3 Brochure, Pickering Laboratories, Mountain View, CA, 1992.
31

Analysis of Amino Acids

Using post-column derivatization via ninhydrin

Column:

Protein Hydrolyzate HighEfficiency (6-m, 150 x 4 mm)

Mobile Phase: Na315 and Na740

Flow Rate:
0.3 mL/min at 55 C
Detection:
Reagent:

Vis at 550 nm
Trione 0.3 mL/min at 130 C

Peak identification
1. Asp
11. Iso
2. Thr
12. Leu
3. Ser
13. Tyr
4. Glu
14. Phe
5. Pro
15. Lys
6. Gly
16. NH3
7. Ala
17. His
8. Cys
18. Trp
9. Val
19. Arg
10. Met
32

Analysis of OPA Precolumn

Derivatives of amino acids

Soy Protein Hydrolyzate

Column:

Pecosphere 3 x3 C18
(3-m, 33 x 4.6 mm i.d.)

Mobile Phase: A: Methanol

B: 1.5% THF in 50 mM
NaOAc (pH 5.9)
Linear gradient of 5% A to
80% A in 6.5 min
Flow Rate:

2.5 mL/min

Detection:

Fluorescence 340 nm (Ex) /

450 nm (Em)

Sample:

Soy protein hydrolyzate

derivatized with OPA

PE Brownlee LC Application Note LCFD-3.

33

Food Additives
Acidulants
Sweeteners - aspartame, saccharin
Flavor compounds
Bitter, pungent, aromatic compounds, and flavor enhancers

Antioxidants
Preservatives
Colors and food dyes
Vitamins - water-soluble and fat-soluble vitamins
Reference: K. Saag, Determination of food additives by HPLC, in HPLC in Food Analysis, R.
Macrae (ed.), Academic Press, London, UK, 1988.

34

Analysis of Sweeteners

and additives in soft drinks

Column:

Pecosphere 5 x 15C C18

(5-m, 150 x 4.6 mm)
Mobile Phase: 45% A in B
A: Methanol
B: Water with 0.7 g/L
hexane sulfonic acid
and H3PO4 to pH 2.1
Flow Rate:
1.5 mL/min
Detection:
UV at 254 nm
Sample:
Filtered, degassed soft drink
1. Matrix
2. Saccharin
3. Caffeine
4. Aspartame
5. Vanillin
6. Sodium Benzoate
PE Brownlee LC Application Note LCFD-6.
35

Bitter Components

Commercial hop extract for brewery (beer)

Column:

C18
(5-m, 250 x 4 mm)
Mobile Phase: 50 - 95% MeOH/1% H3PO4
in 60 min
Flow Rate:
1 mL/min
Detection:
UV at 280 nm
Peak identification:
1 Cohumulinic Acid
2 Humulinic Acid/Cohulupone
3 Hulupone
4 cis/trans-isocohumulone
5,6 cis/trans-isohumulone/Dehydrocohumulinic Acid
7 cis/trans-isoadhumulone
8 Dehydrohumulinic Acid
9 Cohumulone
10 Humulone/Adhumulone
11 Colupulone
12 Lupulone/Adlupulone

36

Phenolic Acids and Flavanols

Extracted from beer with ethyl acetate

Column:

Pecosphere 5 x 15C 18
(5-m, 150 x 4.6 mm i.d.)
Mobile Phase: A: CH3COOH/CH3CN/H2O
(5:45:50)
B: CH3COOH/H20 (3:97)
Linear program from 10% A
to 70% A in 8 min
Flow Rate:
2.5 mL/min
Detection:
UV at 280 nm
1. Gallic Acid
2. 3,4-dihydroxy
Phenylacetic Acid
3. Gentisic Acid
4. Vanillic Acid
5. Caffeic Acid

37

6. Vanillin
7. p-Courmaric Acid
8. Ferulic acid
9. Sinapic Acid
10. Salicylic Acid
11. 5-methoxysalicylic
Acid

HPLC Analysis of Capsaicins

in Hot Sauces

RP-18 Spheri-5 (100 x 1 mm i.d.)

1% HOAC, 40% ACN/H2O,
100 L/min at 35o, 1100 psi
140C pump, LC-235C detector

Capsaicin (C)

mAU (280 nm)

Dihydrocapsaicin (DC)

Calibration Standard
NDC

HC
C
DC

NDC

DC

Minutes
38

HDC

Tobasco Sauce
3200 Scoville Units
Red Hot Sauce
550 Scoville Units

39

Totalchrom Report on Heat Levels

Using customized values

Report: Scoville heat values of Tobasco sauce

40

Time
(min)

Area

5.12
5.76
9.18
12.9

17343
257942
125620
51739

Name

amount
(ng)

NDC
Capsaicin
DHC
HDC

3.37
50.1
24.4
10.0

Total

88.0

Capsaicin
%
0.001
0.013
0.006
0.003

Scoville
(million)

Scoville
unit

9.3
16.1
16.1
8.1

78
2020
984
204

0.022

3286

Phenolic Antioxidants in Foods

Column:

Pecosphere C18
(5-m, 150 x 4.6 mm i.d.)
Mobile Phase: A: 5% Acetic Acid in
Acetonitrile
B: 5% Acetic Acid in Water
Linear gradient from 40 100% A in 6 min
Flow Rate:
2.8 mL/min
Detection:
UV at 280 nm
Sample:
5 L filtered ACN extract of a
chicken bouillon cube

PE Brownlee LC Application Note LCFD-8.

41

1.
2.
3.
4.
5.
6.
7.
8.

Propyl Gallate
THBP
TBHQ
Ionox - 100
BHA
Octyl Gallate
Dodecyl Gallate
BHT

Preservatives

in dog food extract

Column:

Pecosphere 5 C18
(5-m, 150 x 4.6 mm)

Mobile Phase: Methanol/water 40:60

0.2% Perchloric acid
Flow Rate:

1.0 mL/min

Detection:

UV at 210 nm

Sample:

10 L a Filtered Methanol
Extract of 1.5 g of Sample

Peak Identification
1. p-Hydroxybenzoic Acid
2. Sorbic Acid
3. p-Hydroxybenzoic Acid Ethyl Ester
4. p-Hydroxybenzoic Acid Propyl Ester
PE Brownlee LC Application Note LCFD-11.

42

Food Dyes in Beverages

Column:

Pecosphere 5C18
(5-m, 150 x 4.6 mm)
Mobile Phase: Linear Gradient of 30%
Methanol/0.01 M KH2PO4 to
100% MeOH in 4 min
Flow Rate:
3.5 mL/min
Detection:
UV at 290 nm
Sample:
filtered drink samples
FD&C Dyes
1. Green #8
2. Yellow #5
3 Blue #2
4. Red #33
5. Yellow #6
6. Red #40

43

7.
8.
9
10.
11.
12.

Green #3
Blue #1
Red #4
Orange #4
Orange #10
Red #3

Contaminants: Mycotoxins

Aflatoxins - toxic metabolites from molds

B1, B2, G1, G2 (in grains and nuts)
M1, M2 (in diary products)

Ochratoxins - A, B, and C
Fungal metabolites found in maize, wheats or oats

Zearalenone - found in maize

Citrinine - from molds found in grains
Trichothecenes
deoxinivalenol, diacetoxyscirpenol, T2-toxin

Patulin - found in apple juice

Reference: J. Leitao, et al. Determination of mycotoxins in grains and related products, in Food Analysis by
HPLC, L. Nollet (ed.), Marcel Dekker, New York, 1992.

44

Aflatoxins in Peanuts

Peanut Extract

Standards
B2 ,
G2,

10 ppb G1, B1
3 ppb G2, B2

B2,

10 ppb B2,
1, 1 ppb B2

Column:

Pecosphere 3x3 C18

(3-m33 x 4.6 mm)

Mobile Phase: Methanol/Water 48/52

Flow Rate:

1.5 mL/min

Detection:

Fluorescence,
375 nm Ex, 430 Em

Sample:

Peanut Extract with

MeOH/H2O (85:15)
after Solvent
Partitioning and Silica
SPE Cleanup

B2
G2
B2

PE Brownlee LC Application Note LCFD-1.

45

HPLC Analysis

Antimicrobial residues in animal products

Common antimicrobials and their LC detection:
C18 columns are used in most cases
Tetracycline antibiotics
Beta-lactam antibiotics
Polyether antibiotics
Aminoglycoside antibiotics
Macrolide antibiotics
Nitrofurans
Sulfonamides
Quinoxaline 1,4- dioxide

(UV 355 nm)

(UV 210 nm)
(FL)
(FL)
(UV 231 nm)
(UV 362 nm)
(UV 275 nm)
(UV 350 nm)

Sample Preparation - Deproteinization, solvent extraction,

clean-up (SPE), concentration
J. N. A. Botsoglou, Determination of antimicrobial residues in edible animal products, in Food Analysis by
HPLC, L. Nollet (ed.), Marcel Dekker, New York, 1992.

46

HPLC Separation of Antibiotics

Column:

Pecosphere 5 C18
(5-m, 150 x 4.6 mm)

Mobile Phase: 36/63 v/v MeOH/(0.01 M

Phosphate Buffer Adjusted
to pH 7)
Flow Rate:

1 mL/min

Detection:

UV at 220 nm

Peak Identification
1. 6-Aminopenicillin
Acid
2. Ampicillin
3. Penicillin G
4 Oxacillin
5. Cloxacillin
6. Dicloxacillin
Perkin-Elmer LC Application Note LCPH-3.
47

HPLC Analysis of Pesticides

Residues in food

Sample Preparation
Extraction - homogenization, solvent extraction
Cleanup - solvent partitioning, GPC, silica SPE
Derivatization might be needed in some cases

Common pesticide residues

Insecticides - carbamates, organophosphates
Fungicides - benzimidazoles, thiophanates, dithiocarbamates
Herbicides and growth regulators - ureas, phenylureas, triazines, pyridazines,
glyphosate, diquat/paraquat
Reference: R. J. Bushway, "Analysis of pesticide residues in foods by HPLC,
in Food Analysis by HPLC, L. Nollet (ed.), Marcel Dekker, New York, 1992.

48

Sample Pre-fractionation

Of mackerel oil using GPC

High MW Oil Fraction

Column:

Two PLGel 100

10-m Columns

Mobile Phase: THF

Total
Permeation

Flow Rate:

1 mL/min

Detection:

RI

Sample:

Filtered sample of
mackerel oil

Pesticide
Fraction

Reference: Polymer Laboratories GPC Application Notes 119, Amherst, MA.

49

Residual Carbamates in Spinach Extract

Post-column OPA derivatization with fluorescence detection

Column:

Pickering Carbamate
(5 m, 250 x 4.6 mm)
Mobile Phase: 25% - 75% MeOH/H2O
in 26 min
Flow Rate:
1 mL/min at 42 oC
Detection:
Post-column reaction
NaOH/OPA)
Fluorescence
(330/465nm)
Sample:
20 L of spinach extract
prepared by
solid phase extraction

50

Pesticide Analysis with UV Detection

(Drinking water at 100-2000 ppt)

1.
2.
3.
4.
5.

Bentazon
MCPA
Mecoprop
Chloridazon
Desethylatrazine

6. Metoxuron
7. Hexazinone
8. Simazine
9. Cyanazine
10. Methabenz
thiazuron
11. Chlortoluron
12. Atrazine
13. Monolinuron
14. Isoproturon
15. Diuron
16. Metobromuron
17. Metazachlor
18.Sebutylazine
19. Propazine
20. Terbutylazine
21. Linuron
22. Terbutryn
23. Metolachlor
51

Vitamin C analysis using ion pairing agent

<HPLC system>
- Series 200 Quaternary pump
- 5 channel degassor
- Series 200 autosampler
- Series 200 diode array detector w/Integral LINK
<HPLC conditions>
- HPLC column : C18 4.6x250mm(5um)
- mobile phase A : phosphate buffer with tetrabutylammonium hydroxide
B: Acetonitrile
- Gradient : 0min(0%B)-5min(0%B)-10min(5%B)
- Detection : 254 nm
- Flow rate : 1 mL/min
- Injection volume : 10 uL

Page
52 52

< Ascorbic acid >

12 Saponin analysis in Ginseng

* Reprinted from Korean Journal of Medicinal Crop Science

vol.13 p.215-219(2007)

<PerkinElmer Series 200 HPLC >

Series 200 quaternary pump with online degasser
Series 200 autosampler
Column oven(peltier type)
UV/Vis detector
<HPLC conditions>
HPLC Column : C18(4.6x150mm, 5 um)
Column temperature : 35
Detection : 203 nm
Injection volume : 10 uL
Flow rate : 1 mL/min
mobile phase : A : Water B : Acetonitrile
mobile phase condition : 0 min(18% B)-22
min(18%B)32min(30%B)60 min(45%B)-85 min(50%B)

<Panaxadiol
saponins>

<Panaxatriol saponins>

Page
53 53

Carbamate analysis using post column derivatization

Carbamate is used as insecticide.

Oxamyl

Fluorescence detection : Ex 330nm Em 465nm

Reagent A : 0.3 mL/min
Reagent B : 0.3 mL/min
Reactor temperature : 100
Reactor volume : 0.5 mL
Column temperature : 42
Column : 4.6 x 250 mm(P/N 1846250) from Pickering

OPA

Thiofluor

Pinnacle
from Pickering

Pickering 12 standard (2.5 ug/mL)

Page
54 54

Aflatoxin analysis

KFDA regulation requires aflatoxin B1

concentration should be below 2 ppb.

For aflatoxin analysis

Kobra cell (fluorescence detection) is needed.
For sample preparation

Page
55 55

B1

B2

G1

G2

AFLAPREP immunoaffinity column is needed.

Aflatoxin G2, B2 : 0.3 ug/mL

Aflatoxin G1, B1 : 1 ug/mL from Supelco

Flexar

Bottle
Bottletray
traywith
with
integrated
integrated
degassing
degassingand
andSW
SW
comm
link
comm link

Inter-component
Inter-component
drain
drainmanagement
management

Built
Builton
onrugged
rugged
PerkinElmer
PerkinElmerLC
LC
technology,
technology,
recognized
recognizedfor
for
reliability
reliability

56

Tube
Tubemanagement
management
for
streamlined
for streamlined
chromatography
chromatography

Elegant, ergonomic user

interface with
streamlined, consistent
look & footprint

Controlled
Controlledby
byboth
both
Chromera
and
Chromera and
TotalChrom
TotalChrom

Reach for more choices in LC analysis.

Flexars unique ergonomic design

Hidden
Hiddeninter-component
inter-component
drainage
drainagesystem
system
Protects
Protectsthe
theuser
userand
andthe
thesystem/lab
system/lab
from
leak
errors
from leak errors
Plug-and-play;
Plug-and-play;drainage
drainagesystem
system
automatically
interconnects
automatically interconnectswith
with
components
added
to
system
components added to system
Works
Worksbehind
behindthe
thescenes
scenesfreeing
freeing
user
to
focus
on
their
application
user to focus on their application

57

works behind the scenes

Technology basis for Fast-LC

Separation efficiency
factor

van Deemter Curve

Decreased packing particle

size results in:

10m

better efficiency at any velocity

3m

optimum efficiency at higher

velocity (higher throughput)
flatter curve (better H) at higher
velocity

Optimum
value

1.9m
But!

Increased pressure

Mobile phase linear velocity,

(Higher throughput)
Need high pressure capability or
Increase T to reduce viscosity:

We do both!
58

Comparison between conventional LC and Fast LC systems

Analytical LC

Fast LC

Typical analysis time

Typical analysis time

15-20 min

2-8 min

0.5-5 ml/min flow

500-6,000 psi
Quaternary low pressure
blending or binary high pressure
blending gradient

Max 6,000 psi valve

0.5-3 ml/min flow

1,000-10,000 (15,000) psi
Binary high pressure blending gradient

Max 15,000 psi valve

High throughput autosampler

Column Oven

Temperature range 4-150plus

Column particle: sub 3 m

Detectors

20-80 pts/ sec sampling frequency

Pump

Autosampler

59

Temperature range 4-90

Column particles: 5m

5-20 pts/ sec sampling frequency

Example of Fast LC: Mix of Pesticides

Brownlee Acqueous 250x4.6x5um on a Series 200 LC

Brownlee HRes 50x2.1x1.9um on a Series 275 HRes LC

Page
60 60

Soft Drink Sweeteners and Flavors: standards

61

Soft Drink Sweeteners and Flavors: standards

Conventional

Get results faster!

HRes

62

Soft Drink Sweeteners and Flavors in drinks

63

Accelerating throughput with UHPLC chromatography

One additional example of faster LC on a more complex sample (orange peel extract), using high
efficiency 1.9m columns with reduced injection volume

FX-10
** ~2x shorter analysis time **

Conventional

1uL injection at 0.5 mL/min on Anal 50 x

2.1mm 1.9m DB C18

Ambient; 5000psi

3uL injection at 0.5 mL/min on

100 x 4.6 mm 3m C18

Ambient; 2600 psi

64

Thank You

65