Published: Journal of Chromatographic Science, Volume 33, Number 5, May 1995, pp. 273-274.
| Letter to the Editor |
The Correct Expression of the Equivalent Chain Length
L.S. Ettre
To The Editor:
This is in reference to the review paper on reversed-phase high-performance
liquid chromatography (HPLC) of polyunsaturated fatty acids by Rao, Hidajat,
and Ching, published in the January 1995 issue (1). According to this paper,
özcimder and Hammers (2) expressed the so-called equivalent chain length
(ECL) of unsaturated fatty acids in HPLC as
| ECL = N – 2n |
Eq 1
|
where N is the number of carbon atoms in the fatty acid molecule, and n is the number of double bonds in the chain.
The ECL is a term similar to the Kováts retention index, except that while the latter is a general expression, ECL was specifically introduced to express the retention of unsaturated or branched-chain fatty acids. The Kováts index is equal to 100 times the number of carbon atoms in the molecule of a hypothetical n-paraffin, which would have the same retention as the compound of interest; on the other hand, in the case of the ECL, the number of carbon atoms of a hypothetical straight-chain saturated fatty acid with the same retention is given. As indicated by Rao and co-workers, the ECL concept was originally introduced for the gas chromatographic (GC) analysis of (the methyl esters of) fatty acids and was later adopted for HPLC.
There are two major errors in the relationship as given by Rao and co-workers.
The first error is that Equation 1 is given by Rao and co-workers as a general
expression “for HPLC separations,” which it is not; the negative sign
in the right-hand side of the equation is valid only for chromatographic systems
with a nonpolar column, where the unsaturated fatty acid elutes before the saturated
fatty acid with the same carbon number. In other words, in HPLC, the negative
sign is valid only for reversed-phase systems. Similarly, in GC, this is the
situation only for nonpolar stationary phases such as Apiezon grease. For normal-phase
HPLC systems, or for GC analyses with polar stationary phases, the sign is positive,
that is, the corresponding saturated fatty acid (methyl ester) will elute first.
The second error in Equation 1 as given by Rao and co-workers is the coefficient
of n. Nowhere in the paper by özcimder and Hammers (2) is a coefficient
of 2 given; they expressed the relationship for reversed-phase liquid chromatography
as follows:
| ECL = N – yn |
Eq 2
|
emphasizing that y is not a general constant.
If we want to write a generalized relationship for ECL—both for GC and
for HPLC—then it should be given by the following equation:
| ECL = N + (±a)n |
Eq 3
|
where the sign of a is negative for nonpolar GC phases and for reversed-phase HPLC columns and positive for polar GC phases and for normal-phase HPLC columns.
Although Rao and co-workers stated that özcimder and Hammers modified the ECL concept for HPLC, they actually used it only in general terms to explain the retention characteristics of such a system; actual ECL values were determined by GC after prefractionation by reversed-phase HPLC. Rao and co-workers list the ECL values for C20:5 and C22:6 fatty acids as 10; I do not know where they took this value because no reference is given, and it looks as if they simply calculated the values using Equation 1. The paper by özcimder and Hammers lists the ECL values of these two fatty acids determined by GC on polar stationary phases (OV-275 dicyanoallylsilicone and Silar 10C cyanoproplysilicone) as 24.67 and 27.17, respectively. Incidentally, these data would indicate the respective values of 0.93 and 0.86 for a (with a positive sign) in Equation 3 for these acids.
I realize that a review article cannot go into details. However, it should not present incorrect information.
One more remark. Rao and co-workers credit Miwa and co-workers (3) for the introduction of the ECL concept in GC. For historical accuracy, it should be noted that their publication was preceeded by that of Woodford and Van Gent (4) who described the same concept, only using a different name; they called the expression simply the “carbon number”.
References
L.S. Ettre, Ph.D.
Department of Chemical Engineering
Yale University
New Haven, CT 06520-8286
The Authors Reply:
As pointed out by Dr. Ettre, it is true that the equivalent chain length (ECL)
is a term similar to Kováts retention index. In our paper (1), we mentioned
that fatty acid retention in reversed-phase HPLC generally follows the ECL concept.
The concept was originally introduced by Miwa and co-workers (2) as the retention
mechanism of fatty acids in GLC. The same was modified by Özcimder and
Hammers (3) for HPLC separations. Then, we presented the following expression
for ECL:
| ECL = N – 2n |
Eq 1
|
where N is the number of carbon atoms in the fatty acid, and n is the number of double bonds in the chain. This expression is entirely different from that of Özcimder and Hammers (3). Their expression was
| ECL = N – yn |
Eq 2
|
We submit that our expression was taken from Brown and co-workers (4), which
was reference 20 in our paper. We regret our oversight in not mentioning this
reference explicitly immediately after the expression. Further, the negative
sign on the right-hand side was retained in our expression because the review
paper essentially deals with reversed-phase HPLC. Consequently, the ECL values
for C20:5 and C22:6 fatty acids were calculated as 10 from our expression. Finally,
because our paper centered around the concept of ECL, we referred to the work
of Miwa and co-workers (2) as this term was first introduced by them. However,
we thank Dr. Ettre for setting the record straight by pointing out the precedence
of the publication by Woodford and Van Gent (5) who used the same concept albeit
with a different term (carbon number).
References
Mandava S. Rao, K. Hidajat, and C.B. Ching
Department of Chemical Engineering
National University of Singapore
10 Kent Ridge Crescent
Singapore 0511
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