HPLC Preparative Scaleup for Phospholipids
in Soybean Extracts
C. Woodward, R. E. Majors, B. Permar, and Q. Wang
Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808
Goals of This Project
Important Phospholipids in Soybean Extract
Comparison of Mixture of Phospholipid Standards With and Without
Ammonium Acetate in Mobile Phase (Analytical Scalar Column)
Schematic of ESA ELSD
Comparison of Crude Lecitin Extracts on Analytical and Preparative
Columns under Moderately Heavy Loaded Conditions
H exane-IPA -M eO H
1. To develop an acceptable analytical HPLC separation for
soybean phospholipids using a new Agilent scalar silica
gel-based column
ADC1 A, ADC1 CHANNEL A (PHOSLIP2\PLD04600.D)
Norm.
Phospholipid
12000
Abbreviation
A nalyticalScalar
SIL,1 m g loading
11000
10000
2. Demonstrate that a linear scale-up to a new Agilent
Preparative silica gel column can be achieved.
Phosphatidylethanolamine
PE
Phosphatidylserine
PS
9000
8000
7000
5
3. Using automated fraction collection, demonstrate that a
relatively pure fraction from a crude lecithin mixture can be
collected by re-injecting it onto the analytical scalar column
Phosphatidylinositol
PI
Phosphatidylcholine
PC
10
15
20
25
30
H exane-IPA -M eO H + 10 m M N H 4O A c
ADC1 A, ADC1 CHANNEL A (PSTDNAC1\PLSTD464.D)
Both Sets of Date
Obtained Using
Standard
Gradient
Conditions
min
A gilentPrep,
21.2 m g Loading
Norm.
PE
500
4. Demonstrate the usefulness of a modern Evaporative Light
Scattering Detector (ELSD) in detecting phospholipids eluting
from a preparative column at high flow rate
PS + PI
400
PC
300
200
0
100
2
1.
2.
3.
4.
5.
6.
7.
8.
Structure of Typical Phospholipid
Experimental Approach
1.
2.
3.
4.
5.
6.
7.
Chromatographically Important Chemical
Characteristics of Phospholipids
1. They are in class of polar lipids and are amphipathic
2. They contain an ionic portion due to phosphate moiety
3. May contain hydrophilic portion or additional ionic portion due
to presence of choline, serine, ethanolamine, myo-inositol,
and/or glycerol functionality (glycerolphospholipids)
4. They contain a hydrophobic portion due to presence of fatty
acid moiety and complexity results since one or two fatty acids
can be present on the 1,2-diacylglycerol portion.
5. They are generally have low water solubility and can be
extracted with nonpolar solvents
6. They have very low UV absorbance since their absorbance
arises only from the double bond in the fatty acid moiety
Initially, we searched the literature for an analytical method using normal
phase (adsorption) chromatography on silica gel and a solvent system
compatible with ELSD.
A ternary gradient method of Kang and Row [J. Chromatogr.A, 949, 217-223
(2002)] using a hexane-isopropanol-methanol mobile phase was tried
initially with low UV detection.
Since we had a binary preparative pumping system, we attempted to match
as closely as possible the above gradient conditions.
We found severe peak distortion when using these solvents (see following
data)
By the addition of a small amount of ammonium acetate, a volatile additive,
peak shape on both analytical and preparative scale improved dramatically
and the salt was compatible with the ELSD.
We used this method to scale up from the analytical column to a 21.2-mm
I.d. preparative column of the same silica and same particle size (10-um)
We collected fractions of two of the major components to see if we could
enhance the purity of the phospholipids of soybean extract.
12
14
16
18
m in
A gilentPrep-SIL,21.2 x 150m m ,10µ
Split~ 40:1
1000
Fraction:
1
42.4m g on colum n
2
800
600
400
200
0
5
10
15
20
25
m in
mV
400
AgilentPrep-SIL Scalar,4.6 x 150m m ,10µ
350
92% Purity
O riginalPurity,25%
300
(We would kindly like to
Thank ESA for the loan of
The Chromachem Detector)
250
200
150
100
50
5
10
15
20
25
m in
mV
AgilentPrep-SIL Scalar,4.6 x 150m m ,10µ
160
140
75% Purity
O riginalPurity,17%
120
100
80
60
5
10
15
20
25
m in
Conclusions
1.
We have demonstrated the utility of using the Agilent Purification System
with the Chromachem ELSD detector for the gradient analysis of
phospholipids in analytical and preparative columns; a flow splitter prior to
the detector is required for preparative flow rates
2.
We have shown that using a new spherical silica gel sorbent we can
separate a crude lecithin on an analytical (scalar) column and then provide
a good scaleup to a preparative column with the same silica, the same
particles size, and the same length.
3.
We have demonstrated that with a normal phase solvent system (hexaneIPA-methanol), a low amount (10-mM) of ammonium acetate provides
better peak shape than with no salt present
4.
Collected fractions from a crude lecithin preparative run resulted in about a
4-fold improvement in peak purity compared to the crude mix.
AgilentPrep-SIL Scalar,4.6 x 150m m ,10µ
2000
80000
A m ounton colum n:
60000
1500
40000
20000
1000
2.0m g
1.0 m g
0
0.40 m g
0
1
2
3
4
5
500
0.20 m g
M ass injected,m g
0.10 m g
0
0
5
10
15
20
25
30
m in
Crude Lecithin Loadability on Agilent
Prep- SIL Column with ELSD Detection
PI
PC
A gilentPrep-SIL,21.2 x 150m m ,10µ
ADC1 A, ADC1 CHANNEL
CHANNEL AA (PHOSLIP2\PLS04624.D)
(PHOSLIP2\PLS04624.D)
Norm.
ADC1 A, ADC1 CHANNEL A (PHOSLIP2\PLD04622.D)
Flow Split~ 40:1 to ELSD
Norm.
mV
20000
18000
H exane-IPA -M eO H
2500
16000
14000
150
12000
10000
2000
100
8000
6000
5
10
15
20
25
5
30
min
10
15
20
25
30
min
20
25
A m ounton colum n:
1500
Column
42.4 m g
Norm.
Norm.
1400
350
21.2m g
1000
8.48 m g
1200
300
H exane-IPA -M eO H
+ 10 m M N H 4O A c
1000
500
250
4.24 m g
800
200
600
2.12 m g
150
Microsplitter
(Upchurch)
25
1400
1200
mV
200
Waste
20
mV
Detector: Chromachem ELSD,
(ESA, Cambridge, MA)
Temp. Nebulizer: 45 deg.
Temp. Evaporator: 65 deg.
Filter: High
Gas Pressure: 23 psi
Attenuation: 1
250
Pump
B
15
2500
300
Injector
10
Crude Lecithin Fractionation on Agilent PrepSIL with ELSD; Fractions on Scalar Column
Detection
120000
100000
(Initial Experiments using Method of Kang and Row)
Mixer
5
min
Crude Lecithin Loadability on Agilent PrepSIL Scalar Column with ELSD Detection
Effect on Peak Shape of Phospholipids With and Without
Low Concentration of Ammonium Acetate in Mobile Phase
Vapor Exhaust
10
140000
PS
ELSD
A
8
Final Set of Experimental Conditions
ELSD Signal Linearity for Phosphatidylethanolamine
for Analytical Run
Fluid Flow Diagram for Preparative
Chromatography
Pump
Pump
6
Chromatography
Analytical Runs:
Column: Agilent Prep SIL Scalar Column, 4.6- X 150-mm, 10-um
Mobile Phase: A = 94.5:2.5:2.5% hexane-isopropanol-methanol
B = 40:60% isopropanol-methanol
Both solvents contain 10 mM ammonium acetate
Flow Rate: 1.5-mL/min
%B
Gradient: Time, min
0
0
20
47.5
28
100
30
100
30.1
0
35
0
Preparative Runs:
Column: Agilent Prep SIL Column, 21.2- X 150-mm, 10-um
Mobile Phase: same as above
Flow Rate: 31.9=mL/min
Gradient: same as above
Detects compounds based on mass, not optical activity
Nearly universal—responds to all compounds as long as they are
less volatile than the mobile phase
Unlike refractive index detector, can be used with gradient elution
Can be optimized for analyte sensitivity and baseline stability by
adjustment and optimization of evaporation and nebulization
temperatures
Excellent sensitivity for organic mobile phases
However, sensitivity varies with analyte volatility and
Detector linearity may be compound dependent
Since maximum flow rate for ELSD is 4-mL/min, when using high
flow rate in preparative chromatography, flow splitter must be used
before detector
A rea C ounts
1. They are functioning phosphatides in cell membrane and
are found in all biological membranes
2. They are commonly found in animal and plant tissue and
serve as structural components in membranes
3. They play a role in enzyme activation
4. They are important substances in biomedical,
pharmaceutical, and nutrition research
5. Widely used in food and cosmetic industry and industrial
manufacturing (e.g. oils of soybean, palm, rapeseed, etc.
account for 80% of the worldwide plant oil production)
Phosphatidylcholine (PC)
Photo of Agilent Purification
System Next to ESA ELSD
Characteristics of the ELSD for Detection
of Phospholipids
Why Are Phospholipids Important?
4
400
0
100
200
0
0
2
4
6
8
10
12
14
16
18
min
2
4
6
8
10
12
14
5
10
15
20
25
m in