Diamond Analytics Application Note: DA2000-A

8huplnder Slngh,
1
Suprlya S. kanyal,
1
uavld S. !ensen,
2
Andrew L. uadson,
2
MaLLhew 8. Llnford
1

1
ueparLmenL of ChemlsLry and 8lochemlsLry, 8rlgham ?oung unlverslLy, rovo, u1-84602, uSA
2
ulamond AnalyLlcs, 1260 S 1600 W, Crem, u1-84038, uSA

Introduction
The Diamond Analytics Flare core-shell
column offers unique selectivity for an array
of analytes. As a mixed-mode (weak anion
exchange/C
18
) column, it can be made more
hydrophobic or more charged depending on
the pH of the mobile phase.
1
For example, at
pH 2.0, the column has weak anion
exchange properties, making possible the
separation of critical pairs of acidic
herbicides.
2
Working at the other end of the
pH scale (ca. pH 12), the column behaves in
reversed phase mode, separating triazine
herbicides, amphetamines and tricyclic
antidepressants (TCAs).
3-5
The
amphetamines and TCAs are bases that are
best retained on a reversed phase stationary
phase when they are deprotonated, i.e.,
under elevated pH conditions.
The present work is focused on the stability
and reproducibility of the Flare column at
extremes of pH. For the high pH separations
(pH 12), a complex test mixture (Test
Mixture 1, see Figure 1) was prepared that
contains acidic, basic, and neutral analytes.
For the low pH analyses (pH 2), the test
mixture consists of dicamba and 2,4-
dichlorophenoxyacetic acid (2,4-D) (Test
Mixture 2, see Figure 2). During stability
tests, % RSD values were recorded for
various peak parameters, including retention
times (t
R
), retention factors (k), tailing
factors (T
f
), efficiencies (N/m), and
resolutions (R
s
).


























Figure 1. Names and structures of the analytes in
Test Mixture 1.
!"#$%&'()*)+),- /0& 1,/*)+),- %2 ,3" 4)/5%0& 60/+-,)(7 8+/$" 9):"&;9%&"
<%+'50 /, =:,$"5"7 %2 #>
LLhylbenzene
ropazlne
lmlpramlne
AmlLrlpLyllne
norLrlpLyllne
2,4-dlchlorophenoxyaceLlc acld
0 1 2 3 4 5 6
Time (minutes)
1
50






Figure 2. Names and structures of the analytes in
Test Mixture 2.
Experimental
Stability and reproducibility of the Flare
column at high pH
Fifty consecutive injections of Test Mixture
1 were made on the Flare column on Day 1
to test the intra-day variability of the column
(see Separation Conditions 1). After Day 1,
20 injections were made per day for 6
consecutive days under the same conditions
on the same column to determine the inter-
day variability. More than 4000 column void
volumes of the pH 12 mobile phase were
flushed through the column during this test.
Stability and reproducibility of the Flare
column at low pH
Nearly 8000 column void volumes of a pH
2.0 mobile phase at 60 C (see Separation
Conditions 2) were flushed through the
column, during which time Test Mixture 2
was periodically injected to monitor the
column stability.
Determination of column void volume
At pH 2.0 and 12.0, the retention times of
the solvent peak and the unretained
compound (2,4-D) were used to calculate
the column void volume, respectively.
General Separation Conditions
Analytes: All analytes were purchased from
Sigma-Aldrich (St. Louis, MO).
Chromatograph: Waters 1525 Binary
HPLC pump; Breeze 3.30 SPA software.
Column: Diamond Analytics Flare Mixed-
Mode column (4.6 x 33 mm, 4 m)
Injection volume: 5.0 L
Elution: Isocratic
Detection: UV detection at 254 nm
Flow rate: 1.0 mL/min
Separation Conditions 1
To probe column stability at elevated pH
Test Mixture 1: ca. 2 mg/mL each of 2,4-
D, propazine, ethylbenzene, nortriptyline,
imipramine and amitriptyline in
acetonitrile:water (1:1)
Column temperature: 35 C
Mobile Phase: 70:30 premixed solution of
aqueous phosphate buffer (10 mM) at pH
12: acetonitrile, prepared gravimetrically.











Figure 3. Chromatograms corresponding to the 1
st

and 50
th
injections of Test Mixture 1 on the Flare
column at pH 12. Order of elution of analytes: 2,4-D,
propazine, ethylbenzene, nortriptyline, imipramine
and amitriptyline.
Separation Conditions 2
To probe stability at low pH
ulcamba 2,4-dlchlorophenoxyaceLlc
acld
Test Mixture 2: ca. 0.5 mg/mL of 2,4-D
and dicamba in acetonitrile:water (1:1)
Temperature: 60 C
Mobile Phase: 10:90 (v/v) premixed
solution of H
2
O:acetonitrile, with 1.5%
formic acid as additive.

Results and Discussion
Intra-Day Reproducibility at high pH
Figure 3 shows chromatograms of the first
and fiftieth injections of Test Mixture 1 on
day 1 on the Flare column. These
chromatograms overlap extremely well and
represent 940 column void volumes. All of
the analytes showed good peak shapes, i.e.,
for propazine, ethylbenzene, nortriptyline,
imipramine, and amitriptyline, the average
tailing factors over the 50 injections were
1.31, 1.25, 1.79, 1.34, and 1.27,
respectively. % RSD values were calculated
for various peak parameters, including
retention times, retention factors, tailing
factors, efficiencies, and the resolutions
between neighboring peaks, all of which
point to excellent intra-day reproducibility,
i.e., in most cases % RSD values are below
unity (see Table 1).

Table 1. Intra-day reproducibility results;
6
% RSD
values of various peak parameters for the analytes in
Test Mixture 1 over 50 consecutive injections. t
R
=
retention time, k = retention factor, N/m =
plates/meter, R
s
= resolution, T
f
= tailing factor.

Figures 4a and 4b further show that the
retention factors and efficiencies for all the
analytes remain essentially constant over the
50 injections. For the later eluting analytes,
efficiencies range from ca. 50,000 70,000
N/m.









Figure 4. Plots of (a) retention factor (k) vs. number of
injections, and (b) efficiency (N/m) vs. number of
injections for the analytes in Test Mixture 1 over 50
injections at pH 12. (1 = 2,4-D, 2 = Propazine, 3 =
Ethylbenzene, 4 = Nortriptyline, 5 = Imipramine, 6 =
Amitriptyline).
Inter-Day Reproducibility at high pH
To determine the inter-day variability, the
data from the first day (fifty consecutive
injections) and from the next six days
(twenty consecutive injections per day) were
pooled and various peak parameters,
including retention times, retention factors,
tailing factors, efficiencies, and resolutions
Analyte t
R
k N/m R
s
T
f

Propazine 0.60 0.72 0.68 1.66 0.91
Ethyl-benzene 0.60 0.57 0.59 0.30 1.37
Nortriptyline 0.62 0.52 0.73 0.47 2.90
Imipramine 0.60 0.46 1.07 0.59 1.82
Amitriptyline 0.60 0.45 1.55 0.75 1.72
(b)
1
2
3
4
3
6
0 1 2 3 4 3
?)5" @5)0',"7A
Cl
Cl
O
OH
O
CH
3
Dicamba
2,4-dlchlorophenoxyaceuc acld
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2000 4000 6000 8000
!
"
,
"
0
B
%
0

B
5
"

@
5
)
0
'
,
"
7
A

(%+'50 C%)& C%+'5"7
2,4-u
ulcamba
0
0.3
1
1.3
2
2.3
3
3.3
4
0 2000 4000 6000 8000
!
"
,
"
0
B
%
0

2
/
(
,
%
$

@
D
A

<%+'50 C%)& C%+'5"7
2,4-u
ulcamba
were recorded. Table 2 shows % RSD
values for these different peak parameters,
which are generally less than or equal to 5
%, suggesting good inter-day reproducibility
for the Flare column.


Table 2. Inter-day reproducibility results. % RSD
values of various peak parameters for the analytes in
Test Mixture 1 over a span of 7 days (50 injections
on day 1 and 20 injections per day for the following 6
days): t
R
= retention time, k = retention factor, N/m =
plates/meter, R
s
= resolution, T
f
= tailing factor.

Inter-Day Reproducibility at Low pH
Figure 5 shows a separation of Test Mixture
2, using Separation Conditions 2 (at pH 2.0).
7800 column void volumes of the mobile
phase were flushed through the column at 60
C and % RSD values for t
R
and k are given
in Table 3, which correspond to excellent
stability of the column at low pH. Figure 6








Figure 5. Separation of the analytes in Test Mixture
2, using Separation Conditions 2.
shows plots of retention time and retention
factor vs. column void volume, which again
show the stability of the Flare column at low
pH.




Table 3: % RSD values for retention time (t
R
) and
retention factors (k) of 2,4-D and dicamba for
passage of 7800 column void volumes of mobile
phase through the Flare column using Separation
Conditions 2.

















Figure 6. Plots of (a) retention time and (b) retention
factor vs. column void volumes for 2,4-D and
dicamba at pH 2 on the Flare column.
Analyte t
R
k N/m R
s
T
f

Propazine 1.48 4.08 0.88 4.80 1.35
Ethyl-benzene 1.10 2.68 1.75 1.65 3.41
Nortriptyline 1.42 2.87 1.65 1.42 5.15
Imipramine 1.65 2.96 2.88 1.16 4.23
Amitriptyline 1.69 3.10 2.97 0.79 4.62
Analyte t
R
k
2,4-D 0.62 1.28
Dicamba 0.88 1.26
(b)
(a)

References
1. C. P. Pung, A. A. kazarlan, A. L. uadson, 8.
aull, . n. nesLerenko and M. 8. Llnford, (ulamond
AnalyLlcs, hLLp://dlamond-
analyLlcs.com/uploads/ulamond20AnalyLlcs20Ap
pllcaLlon20Culdellnes.pdf, 2013).
2. 8. Slngh, u. S. !ensen, A. !. Mlles, A. L.
uadson and M. 8. Llnford, (ulamond AnalyLlcs,
hLLp://dlamond-
analyLlcs.com/uploads/Acldlc20Perblcldes.pdf,
2013).
3. L. A. WlesL, u. S. !ensen, A. !. Mlles, A.
uadson and M. 8. Llnford, (ulamond AnalyLlcs,
hLLp://dlamond-analyLlcs.com/uploads/CL822-
AgonlsLs20and20AmpheLamlnes.pdf, 2013).
4. L. A. WlesL, u. S. !ensen, A. !. Mlles, A. L.
uadson and M. 8. Llnford, (ulamond AnalyLlcs,
hLLp://dlamond-
analyLlcs.com/uploads/llareMlxedModeColumn-
1rlazlnePerblcldes.pdf, 2013).
3. u. AnalyLlcs, (ulamond AnalyLlcs,
hLLp://dlamond-
analyLlcs.com/uploads/1rlcycllc20AnLldepressanLs
20(1CAs)1.pdf, 2013).
6. eak parameLers for 2,4-u were noL lncluded ln
1ables 1 and 2 because: (l) an lmpurlLy ln Lhe LesL
mlxLure prevenLed a sLralghLforward deLermlnaLlon
of Lhe 1
f
of 2,4-u and (ll) aL elevaLed pP, 2,4-u ls
deproLonaLed and poorly reLalned on Lhe llare
column so lL co-eluLes wlLh Lhe dead Llme marker,
maklng deLermlnaLlon of efflclency dlfflculL.