0045E Fatty Acid Analysis

Application
Note #0045E
Evaporative Light Scattering Detection
Simplifies Fatty Acid Analysis
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Introduction
A variety of chromatographic
methods have been used to analyze
fatty acids. Poor resolution and
sample decomposition are problems
associated with GC methods [1] .
Detection of underivatized fatty
acids using traditional HPLC
methodology is neither sensitive
nor selective because these
compounds generally do not
contain suitable chromophores. A
number of different derivatization
techniques have been employed to
overcome these obstacles [2,3] ,
2859
however, derivatization often
increases analysis time and
complicates the method.
Evaporative Light-Scattering Detection (ELSD) offers several
advantages over traditional techniques for analyzing fatty acids. Since
the ELSD’s response does not depend on the sample’s optical
characteristics, there is no need for post-column derivatization. This
greatly minimizes sample preparation and removes complications
associated with detection in the far UV (220nm or lower) range. The
ELSD is also compatible with multisolvent gradients for improved
resolution and faster separations.
The objective of this study was to compare traditional UV detection
of free fatty acids to Evaporative Light Scattering Detection, and to
evaluate the separation and sensitivity obtained by a narrow-bore
(2.1mm) and an analytical-bore (4.6mm) column.
Experimental Conditions
The HPLC system consisted of an Alltech (Deerfield, IL) Model 525 Pump
(binary gradient), an Alltech Model 570 Autosampler with a 20µL loop,
an Alltech Model 500 ELSD, a Linear (Thermo Separation Products, San
Jose, CA) 205 UV-Vis Detector set at 210nm, an LC Packings (San
Francisco, CA) Acurate Microflow Processor (flow splitter) set at a 1:20
split ratio, and PE Nelson (San Jose, CA) Turbochrom EL data
acquisition software. Alltech Alltima C18-LL, 5µm columns (250 x
2.1mm and 250 x 4.6mm ID) were used for the separations. Gradient
elution with water and acetonitrile was used.
HPLC-grade solvents and water were purchased from Burdick and
Jackson (Muskegon, MI). Free fatty acid standards were obtained from
Alltech Associates, Inc. (Deerfield, IL). All standards were prepared in
ethanol, and all dilutions of the stock solution were made in 77%
acetonitrile.
Results and Discussion
UV vs. ELSD
To compare the detection sensitivity of underivatized fatty acids with
UV and ELSD, a mixture of six fatty acids was injected onto a 2.1mm ID
column. The UV and ELSD detectors were connected in series. As
shown in Figure 1, the ELSD gives much greater sensitivity than the UV
detector. Since the ELSD’s response does not rely on the optical
characteristics of the sample, it gives a more accurate representation of
sample composition. For example, palmitic acid is one of the most
concentrated of the six fatty acids and yet it is barely visible on the UV
chromatogram.
Narrow Bore vs. Analytical Bore Columns
Operating temperature is a limiting factor when analyzing semi-volatile
analytes with ELSD. When coupled with narrow bore columns, the
detector sensitivity is increased because it can be operated at much
lower temperatures due to lower mobile phase flowrates. In this study,
the 2.1mm ID column achieved the greatest sensitivity. Since sample
dilution decreases with column diameter, sample analytes elute from a
2.1mm ID column at a higher concentration, producing a larger
response from the detector.
In applications where an analytical bore column must be used, a
flow splitter may be placed between the column and the detector to
divert most of the mobile phase to waste before entering the detector.
Figure 2 compares the separation of the same six fatty acids on an
analytical bore column with and without a flow splitter. The flow
splitter reduced the drift tube temperature by 55°C, which eliminated
sample evaporation. This is most significant with myristic acid which
has the smallest carbon number (C14). Even after a 1:20 split, the peak
height of myristic acid is over five times greater with the lower
detection temperature. Also, nearly equivalent response factors are
achieved when sample loss by evaporation does not occur.
Conclusion
The Evaporative Light Scattering Detector (ELSD) coupled with an
Alltima C18-LL, 5µm column provides an excellent alternative to
traditional methods of fatty acid analysis. Good results are obtained
without complicated derivatization techniques associated with GC and
UV detection. The 2.1mm ID column’s low flowrate allows a lower drift
tube temperature which decreases sample evaporation and maximizes
sensitivity. For those who cannot switch to a 2.1mm ID column, the
4.6mm ID column combined with a flow splitter also outperforms
traditional fatty acid analysis methods.
References*
1. D. Marini, In Food Analysis By HPLC (Leo M. L. Nollet, ed.), New York,
New York, p. 176 (1992).
2. L.A. Stenson, In Chemical Derivatization in Analytical Chemistry , Vol.1
(R.W. Freiand and J.F. Lawrence, eds.), Plenum, New York, p. 127
(1981).
3. Van der Wal Sj, J. Liq. Chromatogr., 6:37 (1983).
*Please contact your technical library for references. References are
not available through Alltech.
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Page 1 0045E
© Copyright 2000 Alltech Associates, Inc. Contact your Alltech office or distributor for current or local prices.

Fatty Acids (Underivatized)
1
3
4
5
6
1. Linolenic Acid - 17.9mg/L
2. Myristic Acid - 79.4mg/L
3. Linoleic Acid - 16.9mg/L
4. Palmitic Acid - 51.1mg/L
5. Oleic Acid - 36.2mg/L
6. Stearic Acid - 49.4mg/L
8418
8419
1
2
2 3 45 6
500 ELSD
65°C Drift Tube Temperature
1.50 SLPM Nitrogen Flow
UV at 210nm
0
5 10 15 20 Min.
Column: Alltech Alltima C18-LL, 5µm, 250 x 2.1mm
Mobile Phase: A: Water
B: Acetonitrile
Gradient: Time: 0 10 15 20
%B: 77 80 80 95
Flowrate: 0.4mL/min
Figure 1 - The 500 ELSD greatly improves detection sensitivity compared to UV in the analysis of free fatty acids.
Fatty Acids (Underivatized)
0
1
2
3
12 5
3
4
4
Column: Alltech Alltima C18-LL, 5µm, 250 x 4.6mm
Mobile Phase: A: Water
B: Acetonitrile
Gradient: Time: 0 10 15 20
%B: 77 80 80 95
Flowrate: 2.0mL/min
Detector: 500 ELSD
5
5 10 15 20 Min.
6
6
1. Linolenic Acid - 601.8mg/L
2. Myristic Acid - 660.6mg/L
3. Linoleic Acid - 554.4mg/L
4. Palmitic Acid - 606.6mg/L
5. Oleic Acid - 579.0mg/L
6. Stearic Acid - 609.0mg/L
With Flow Splitter
30°C Drift Tube Temperature
2.00L/min Nitrogen Flow
flow splitter (by LC Packings) placed between
column and detector 1:20 Split Ratio
Without Flow Splitter
85°C Drift Tube Temperature
3.75L/min Nitrogen Flow
8420
8421
Figure 2 - A flow splitter in-line prevents sample evaporation and provides nearly equivalent response.
Columns
DESCRIPTION
PART NO.
Alltech Alltima C18-LL Column, 5µm
4.6 x 250mm 88099
2.1 x 250mm 88389
6/05/00 Page 2 0045E
© Copyright 2000 Alltech Associates, Inc. Contact your Alltech office or distributor for current or local prices.