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Poster Note LPN 2880 from HPLC 2011
2014-05-12T18:05:59.903-04:00
Emma Ramirez
Impact of Solvent Mixing on Liquid Chromatographic Performance
2012-04-02T11:23:35-04:00
2012-04-02T11:23:02-04:00
2012-04-02T11:23:35-04:00
Microsoft PowerPoint
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2014-05-12T17:01:15.523-04:00
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Emma Ramirez
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17.7257 0 0 17.7256 1648.645 2245.6714 Tm
(The )Tj
2.001 0 Td
(UltiMate)Tj
11.8172 0 0 11.8171 1749.1226 2250.989 Tm
(\231)Tj
17.7257 0 0 17.7256 1760.9397 2245.6714 Tm
( 3000 Binary RSLC System consisted of the following modules: )Tj
/C2_0 1 Tf
-6.335 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(SRD-3400 Integrated Solvent and Degasser Rack, Four Channels )Tj
/C2_0 1 Tf
-0.722 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(HPG-3200RS Binary Rapid Separation Pump )Tj
/C2_0 1 Tf
-0.722 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
[(WPS-3000TRS Rapid Separation W)18(ell Plate Sampler)55(, )]TJ
24.364 0 Td
(Thermostatted)Tj
( )Tj
/C2_0 1 Tf
-25.086 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
0.26 0 Td
(TCC-3000RS Rapid Separation )Tj
14.267 0 Td
(Thermostatted)Tj
( Column Compartment )Tj
/C2_0 1 Tf
-15.249 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
[(VWD-3400RS Rapid Separation Four-Channel V)74(ariable )]TJ
0 -1.167 TD
( )Tj
[(W)37(avelength Detector )]TJ
/C2_0 1 Tf
-0.722 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(Semi-Micro Flow Cell, 2.5 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(L, Stainless Steel )Tj
/C2_0 1 Tf
-0.722 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
0.26 0 Td
[(Thermo Scientific Dionex V)18(iper System Capillaries )]TJ
-0.982 -1.222 Td
( )Tj
0 -1.333 TD
(The )Tj
2.001 0 Td
(UltiMate)Tj
( 3000 Quaternary RSLC System consisting of the following )Tj
-2.001 -1.167 Td
(modules: )Tj
/C2_0 1 Tf
0 -1.667 TD
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(SR-3000 Integrated Solvent and Degasser Rack, Four Channels )Tj
/C2_0 1 Tf
-0.722 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(LPG-3400RS Quaternary Rapid Separation Pump )Tj
/C2_0 1 Tf
-0.722 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
[(WPS-3000TRS Rapid Separation W)18(ell Plate Sampler)55(, )]TJ
24.364 0 Td
(Thermostatted)Tj
( )Tj
/C2_0 1 Tf
-25.086 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
0.26 0 Td
(TCC-3000RS Rapid Separation )Tj
14.267 0 Td
(Thermostatted)Tj
( Column Compartment )Tj
/C2_0 1 Tf
-15.249 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
[(VWD-3400RS Rapid Separation Four-Channel V)74(ariable )]TJ
0 -1.222 TD
( )Tj
[(W)37(avelength Detector )]TJ
/C2_0 1 Tf
-0.722 -1.611 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
(Semi-Micro Flow Cell, 2.5 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(L, Stainless Steel )Tj
/C2_0 1 Tf
-0.722 -1.667 Td
<004D>Tj
/TT0 1 Tf
0.722 0 Td
( )Tj
[(V)18(iper)]TJ
11.8172 0 0 11.8171 1707.4308 1734.9769 Tm
(\231)Tj
17.7257 0 0 17.7256 1719.2479 1729.6593 Tm
( System Capillaries )Tj
0.662 1 0.232 0.326 k
/TT1 1 Tf
29.5429 0 0 29.5427 211.3829 2854.3643 Tm
(Introduction )Tj
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
17.7257 0 0 17.7256 211.3829 2822.8523 Tm
(Gradient elution, e.g., increasing the over analysis time, is currently \
the most )Tj
0 -1.167 TD
[(common technique in reversed-phase \(RP\)-HPLC. )18(There are two dif)18(ferent )]TJ
0 -1.222 TD
(technical solutions for online \(dynamic\) gradient formation: high-pres\
sure )Tj
0 -1.167 TD
(gradient proportioning \(HPG\) and low-pressure gradient proportioning \(\
LPG\). )Tj
0 -1.222 TD
(In both solutions, compositional fluctuation occurs during gradient form\
ation, )Tj
0 -1.222 TD
[(causing a deterioration of chromatographic performance. )18(This can be reduced )]TJ
0 -1.167 TD
[(with the use of a highly ef)18(ficient mixer to homogenize the mobile phase )]TJ
0 -1.222 TD
(composition. Here, the experimental determination of mixer performance i\
s )Tj
0 -1.167 TD
(described and the impact on liquid chromatographic performance is discus\
sed )Tj
0 -1.222 TD
(for two selected applications using ion-pairing agent. )Tj
15.7562 0 0 15.7561 211.3829 2609.7515 Tm
( )Tj
0.662 1 0.232 0.326 k
/TT1 1 Tf
29.5429 0 0 29.5427 211.3829 2562.8765 Tm
(Experimental Determination of )Tj
0 -1.2 TD
(Mixer Performance )Tj
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
17.7257 0 0 17.7256 211.3829 2495.9133 Tm
(Compositional fluctuations were simulated by a sinus-like gradient oscil\
lating )Tj
0 -1.222 TD
[(between 10\22670% B. )18(The gradient was programmed using the )18(Thermo )]TJ
0 -1.222 TD
(Scientific Dionex )Tj
7.67 0 Td
(Chromeleon)Tj
( Chromatography Data System and performed )Tj
-7.67 -1.167 Td
[(using a )18(Thermo Scientific Dionex )]TJ
14.877 0 Td
(UltiMate)Tj
( 3000 Binary Rapid Separation LC )Tj
-14.877 -1.222 Td
[(\(RSLC\) system. Figure 1 illustrates the observed UV)55(-detector signals with and )]TJ
0 -1.167 TD
[(without a mixer)55(. )18(The mixer performance is determined as the percentage of )]TJ
0 -1.222 TD
(signal attenuation compared to the same instrument without any mixer as \
the )Tj
0 -1.222 TD
[(reference. )18(The frequency of the sinus-like gradient multiplied by the flow rate )]TJ
0 -1.167 TD
(gives the simulated volume period. For the characterization of UHPLC mix\
ers )Tj
0 -1.222 TD
(with a typical dwell volume of 30\226150 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(L, a volume period of 20 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
[(L)37( was )]TJ
0 -1.167 TD
(chosen. For the characterization of mixers with dwell volumes larger tha\
n )Tj
0 -1.222 TD
(400 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
[(L)37( \(still the standard for conventional HPLC systems\), the experiments )]TJ
0 -1.167 TD
(were performed with a volume of 200 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(L. For an objective and precise )Tj
0 -1.222 TD
(comparison of mixer performances, the mixer dwell volume was )Tj
0 -1.222 TD
[(experimentally determined according to Dolan and Snyder)55(.)]TJ
11.8172 0 0 11.8171 671.5336 2202.8501 Tm
(1 )Tj
0.662 1 0.232 0.326 k
23.6343 0 0 23.6342 211.3829 2170.9436 Tm
( )Tj
0.75 0.68 0.655 0.902 k
/TT1 1 Tf
17.7257 0 0 17.7256 211.3829 2097.7495 Tm
(FIGURE 1)Tj
[(. UV)55(-detector absorbance signal of the sinus-like gradient with )]TJ
0 -1.167 TD
[(\(red\) and without \(black\) a mixer)55(. The signal attenuation quantifies the )]TJ
0 -1.222 TD
(mixer performance)Tj
/TT2 1 Tf
(. )Tj
0.662 1 0.232 0.326 k
/TT1 1 Tf
29.5429 0 0 29.5427 213.0652 881.1674 Tm
[(On-Column )37(Amplification of Mixing Ripple)]TJ
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
20.017 0 Td
( )Tj
17.7257 0 0 17.7256 213.0652 849.6552 Tm
(Ion-pairing agents are widely used for RP-HPLC to manipulate the pH and \
the )Tj
0 -1.167 TD
(interaction of the )Tj
(analytes)Tj
( with the stationary phase in order to enhance )Tj
0 -1.222 TD
(separation. )Tj
5.208 0 Td
[(T)37(rifluoroacetic)]TJ
[( acid \(TF)55(A\) is the most common ion-pairing agent )]TJ
-5.208 -1.167 Td
[(used for peptide and protein separations. Unfortunately)74(, )18(TF)55(A)55( also causes some )]TJ
0 -1.222 TD
[(undesirable ef)18(fects. )18(The absorbance of )18(TF)55(A)55( below 250 nm dramatically )]TJ
0 -1.222 TD
[(changes depending on the water/acetonitrile ratio. )18(This causes a strong )]TJ
0 -1.167 TD
[(baseline shift during gradient elution. )55(A)55( compensation of this ef)18(fect is possible if )]TJ
0 -1.222 TD
[(the )18(TF)55(A)55( concentration in acetonitrile is approximately 15% lower than in the )]TJ
0 -1.167 TD
(aqueous solvent. )Tj
0 -1.911 TD
[(The other side ef)18(fect results from the retentive interaction of )18(TF)55(A)55( with RP)18( )]TJ
0 -1.2 TD
[(columns. )18(Therefore, the )18(TF)55(A)55( concentration of the mobile phase stream behind )]TJ
0 -1.222 TD
(the column fluctuates with varying organic solvent concentration. In cas\
e of )Tj
0 -1.167 TD
[(incompletely mixed or fluctuating mobile phase content, the dynamic )18(TF)55(A)55( )]TJ
0 -1.222 TD
[(equilibrium on the column is disturbed. )18(This causes a strong amplification of )]TJ
0 -1.167 TD
[(mixing ripples by the column. Since )18(TF)55(A)55( absorbs UV light considerably )]TJ
0 -1.222 TD
(stronger than water or acetonitrile, significant baseline ripples are ob\
served.)Tj
11.8172 0 0 11.8171 807.1976 524.0944 Tm
(2)Tj
17.7257 0 0 17.7256 813.7697 518.7767 Tm
( )Tj
-33.889 -1.222 Td
(This results in a decreasing signal-to-noise ratio, which significantly \
reduces )Tj
0 -1.167 TD
(the limit of detection \(LOD\) and limit of quantification \(LOQ\). )Tj
19.6953 0 0 19.6951 213.0652 462.6457 Tm
( )Tj
17.7257 0 0 17.7256 213.0652 442.9504 Tm
[(T)111(o quantify the amplification ef)18(fect of the column on the baseline ripple, two )]TJ
0 -1.167 TD
(sets of experiments were performed. In the first set, the baseline rippl\
es were )Tj
0 -1.222 TD
[(determined without a column. )55(A)55( 50 )]TJ
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(m )Tj
17.252 0 Td
(i.d.)Tj
1.334 0 Td
( fused-silica capillary was used as )Tj
-18.586 -1.222 Td
(a restrictor to generate a back-pressure of40 )Tj
20.01 0 Td
(MPa)Tj
(. In the second set of )Tj
-20.01 -1.167 Td
(experiments, the baseline ripples were determined using a column )Tj
T*
[(\(Thermo Scientific )55(Acclaim 120 C18, 3 )]TJ
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(m, 120 )Tj
21.14 0 Td
(\305)Tj
(, 250 )Tj
/C2_2 1 Tf
3.169 0 Td
<0001>Tj
/TT0 1 Tf
( 3.0 mm\). For a )Tj
-24.309 -1.167 Td
(reproducible and evaluable determination of baseline ripples, each run s\
tarted )Tj
0 -1.222 TD
[(with an isocratic elution of 5% B \(dial-a-mix\) for five minutes. )18(The total )]TJ
0 -1.167 TD
[(absorption dif)18(ference between 100% )55(A)55( and 100% B was 160 )]TJ
27.057 0 Td
(mAU)Tj
(. Figure 3 )Tj
-27.057 -1.222 Td
(depicts the baseline with and without column. )Tj
0 -1.911 TD
( )Tj
19.6953 0 0 19.6951 931.7371 2391.019 Tm
(Plotting the mixer performance against the remaining baseline ripple )Tj
0 -1.2 TD
(roughly shows an exponential decay curve as already found in the )Tj
0 -1.2 TD
(previous experiments. It also clearly illustrates the strong amplifying \
)Tj
0 -1.2 TD
(behavior of the column. Figure 4 shows the comparison between the )Tj
T*
(baseline ripple with column and the baseline ripple with a restrictor )Tj
0 -1.2 TD
[(capillary)74(. )18(The amplification of the baseline ripple varies between 37 and )]TJ
0 -1.2 TD
(58 depending on the mixer performance, with an average of 44, as )Tj
0 -1.3 TD
(shown in Figure 5)Tj
21.6648 0 0 21.6647 1087.1931 2223.6104 Tm
(. )Tj
23.6343 0 0 23.6342 931.7371 2181.8562 Tm
( )Tj
11.8172 0 0 11.8171 1648.645 366.8681 Tm
(SecurityGuard)Tj
( is a trademark of )Tj
(Phenomenex)Tj
(, Inc. )Tj
22.678 0 Td
[(All other trademarks are the property of )18(Thermo Fisher Scientific )]TJ
-22.678 -1.167 Td
(and its subsidiaries. )Tj
0 -1.767 TD
(This information is not intended to encourage use of these products in a\
ny manners that might infringe the intellectual )Tj
0 -1.233 TD
(property rights of others. )Tj
0 -1.767 TD
(LPN2880-02 )Tj
17.7257 0 0 17.7256 211.3829 1575.6354 Tm
(The plot of the mixer dwell volume against the remaining baseline ripple\
)Tj
0 -1.167 TD
(matches an exponential decay curve quite well, e.g., the one found in )Tj
0 -1.222 TD
[(discharge of a capacitor)55(. Figure 2 illustrates the decay curve for the two )]TJ
0 -1.167 TD
(volume periods of 20 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
[(L)37( and 200 )]TJ
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
[(L. )18(The volume period for this set of )]TJ
0 -1.222 TD
(experiments shows the typical behavior of a decay constant, )Tj
/C2_2 1 Tf
26.959 0 Td
<0002>Tj
/TT0 1 Tf
(. )Tj
/TT1 1 Tf
13.84 77.585 Td
(FIGURE 3. )Tj
(Observed baseline with \()Tj
0.81 0.642 0 0 k
(blue)Tj
0.75 0.68 0.655 0.902 k
(\) and without \()Tj
0 1 1 0 k
(red)Tj
0.75 0.68 0.655 0.902 k
(\) column. )Tj
53.1772 0 0 53.1769 211.3829 3236.7954 Tm
(Impact of Solvent Mixing on Liquid Chromatographic Performance )Tj
39.3905 0 0 39.3903 211.3829 3158.4087 Tm
(Christian Schmidt, )Tj
[(V)55(erena)]TJ
12.393 0 Td
( )Tj
(Jendreizik)Tj
(, )Tj
[(W)18(ulff)]TJ
( )Tj
(Niedner)Tj
12.151 0 Td
(, and Fraser McLeod)Tj
/TT2 1 Tf
( )Tj
/TT1 1 Tf
-24.543 -1.325 Td
(Thermo Fisher Scientific, )Tj
(Germering)Tj
17.282 0 Td
(, Germany )Tj
ET
q
628.5660248 0 0 398.6533661 211.3829041 1628.9870148 cm
/Im61 Do
Q
q
628.1859741 0 0 398.4122925 210.0287933 971.7072144 cm
/Im62 Do
Q
BT
/TT1 1 Tf
17.7257 0 0 17.7256 213.065 1409.7305 Tm
(FIGURE 2. Exponential decay-curve behavior of remaining baseline )Tj
0 -1.167 TD
(ripples as a function of the mixer volume. )Tj
ET
q
624.6229706 0 0 396.1525879 931.7371826 2444.4260254 cm
/Im63 Do
Q
q
624.1812592 0 0 395.872406 931.7371826 1735.5580139 cm
/Im64 Do
Q
BT
/TT1 1 Tf
17.7257 0 0 17.7256 931.7371 2167.9604 Tm
(FIGURE 4)Tj
(. Influence of the mixer volume on the remaining baseline ripple )Tj
0 -1.167 TD
(with \()Tj
0 1 1 0 k
(red)Tj
0.75 0.68 0.655 0.902 k
(\) and without \()Tj
0.855 0.66 0 0 k
(blue)Tj
0.75 0.68 0.655 0.902 k
(\) a column. )Tj
ET
q
624.6228485 0 0 396.1524506 931.7371826 1232.109024 cm
/Im65 Do
Q
BT
/TT1 1 Tf
17.7257 0 0 17.7256 931.7371 1663.4484 Tm
(FIGURE 5)Tj
[(. )37(Amplification in baseline ripple by the column as a function of )]TJ
T*
(the mixer volume. )Tj
0.662 1 0.232 0.326 k
29.5429 0 0 29.5427 934.5682 1156.8962 Tm
(Mixing Ripple Induced Peak Shape )Tj
0 -1.2 TD
(Distortion)Tj
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
( )Tj
17.7257 0 0 17.7256 934.5682 1089.9324 Tm
[(The retention mechanism of RP-HPLC dif)18(fers between small and large )]TJ
0 -1.167 TD
[(molecules. Because of the higher adsorption enthalpy)74(, proteins are adsorbed )]TJ
0 -1.222 TD
(onto the hydrophobic surface of the column and remain there until the )Tj
0 -1.167 TD
(concentration of the organic modifier is high enough to elute the molecu\
les )Tj
0 -1.222 TD
[(from the hydrophobic surface. )18(The elution order is related to the hydrophobic )]TJ
0 -1.222 TD
(nature; the more hydrophilic the proteins, the earlier it will be eluted\
from the )Tj
0 -1.167 TD
[(hydrophobic surface under gradient conditions. )18(This concentration-)]TJ
29.555 0 Td
(dependant)Tj
4.727 0 Td
( )Tj
-34.282 -1.222 Td
(adsorption/desorption process makes the application sensitive to slight \
)Tj
0 -1.167 TD
(deviations in the solvent composition. On the critical concentration ran\
ge of the )Tj
0 -1.222 TD
[(organic modifier)55(, fluctuation can cause partial elution of proteins followed by )]TJ
0 -1.222 TD
(re-adsorption on a later point in the column, thus resulting in distorte\
d peak )Tj
0 -1.167 TD
(shapes.)Tj
11.8172 0 0 11.8171 996.6516 861.8627 Tm
(3)Tj
17.7257 0 0 17.7256 1003.2237 856.545 Tm
[( With shallow gradients and short columns, this ef)18(fect is more )]TJ
-3.873 -1.222 Td
(pronounced. )Tj
0 -1.722 TD
(Figure 6 shows the expected peak distortion for lysozyme if a small mixi\
ng )Tj
0 -1.222 TD
(volume is applied, although no baseline ripple is visible. Regular peak \
shapes )Tj
T*
[(are observed after changing to a larger mixer)55(. Furthermore, the distorted peak )]TJ
0 -1.167 TD
[(shape has a negative impact on the repeatability)74(. Figure 7 shows the influence )]TJ
0 -1.222 TD
(of the mixer volume on peak height precision and peak width \(50%\) prec\
ision. )Tj
19.6953 0 0 19.6951 934.5682 686.1819 Tm
( )Tj
ET
q
628.158844 0 0 398.3950653 931.7371826 183.1708527 cm
/Im66 Do
Q
BT
/TT1 1 Tf
17.7257 0 0 17.7256 931.7371 624.4498 Tm
(FIGURE 6. Observed peak shapes of lysozyme with small \()Tj
0 1 1 0 k
(red)Tj
0.75 0.68 0.655 0.902 k
(\) and large )Tj
0 -1.167 TD
(\()Tj
0.855 0.66 0 0 k
(blue)Tj
0.75 0.68 0.655 0.902 k
(\) mixer volume. )Tj
ET
q
626.6931152 0 0 397.4654999 1648.6450195 2423.1570587 cm
/Im67 Do
Q
BT
/TT1 1 Tf
17.7257 0 0 17.7256 1648.645 2866.1814 Tm
(Figure 7. Influence of the mixer volume on peak height \()Tj
0.855 0.66 0 0 k
(blue)Tj
0.75 0.68 0.655 0.902 k
(\) and peak )Tj
0 -1.167 TD
(width \(50%, )Tj
0 1 1 0 k
(red)Tj
0.75 0.68 0.655 0.902 k
(\) precision for six replicates. )Tj
0.662 1 0.232 0.326 k
29.5429 0 0 29.5427 1648.645 2344.5762 Tm
(Chromatographic Conditions and )Tj
0 -1.2 TD
(Equipment )Tj
ET
0.307 0.063 0 0 k
1648.645 1560.65 105.493 92.173 re
f*
1754.137 1560.65 169.379 92.173 re
f*
1923.517 1560.65 167.41 92.173 re
f*
2090.926 1560.65 183.658 92.173 re
f*
0.065 0.046 0.047 0 k
1648.645 1504.388 105.493 56.262 re
f*
1754.137 1504.388 169.379 56.262 re
f*
1923.517 1504.388 167.41 56.262 re
f*
2090.926 1504.388 183.658 56.262 re
f*
0 0 0 0 k
1648.645 1448.126 105.493 56.262 re
f*
1754.137 1448.126 169.379 56.262 re
f*
1923.517 1448.126 167.41 56.262 re
f*
2090.926 1448.126 183.658 56.262 re
f*
0.065 0.046 0.047 0 k
1648.645 1391.864 105.493 56.262 re
f*
1754.137 1391.864 169.379 56.262 re
f*
1923.517 1391.864 167.41 56.262 re
f*
2090.926 1391.864 183.658 56.262 re
f*
0 0 0 0 k
1648.645 1335.602 105.493 56.262 re
f*
1754.137 1335.602 169.379 56.262 re
f*
1923.517 1335.602 167.41 56.262 re
f*
2090.926 1335.602 183.658 56.262 re
f*
0.065 0.046 0.047 0 k
1648.645 1271.79 105.493 63.812 re
f*
1754.137 1271.79 169.379 63.812 re
f*
1923.517 1271.79 167.41 63.812 re
f*
2090.926 1271.79 183.658 63.812 re
f*
0 0 0 0 k
1648.645 1215.528 105.493 56.262 re
f*
1754.137 1215.528 169.379 56.262 re
f*
1923.517 1215.528 167.41 56.262 re
f*
2090.926 1215.528 183.658 56.262 re
f*
0.065 0.046 0.047 0 k
1648.645 1159.266 105.493 56.262 re
f*
1754.137 1159.266 169.379 56.262 re
f*
1923.517 1159.266 167.41 56.262 re
f*
2090.926 1159.266 183.658 56.262 re
f*
0.75 0.68 0.655 0.902 K
q 1 0 0 1 1648.645 1560.6499 cm
0 0 m
625.94 0 l
S
Q
q 1 0 0 1 1648.645 1652.8231 cm
0 0 m
625.94 0 l
S
Q
q 1 0 0 1 1648.645 1159.2661 cm
0 0 m
625.94 0 l
S
Q
BT
0.75 0.68 0.655 0.902 k
/TT1 1 Tf
17.7257 0 0 17.7256 1783.363 1631.5524 Tm
(Experimental )Tj
-0.221 -1.167 Td
(Determination )Tj
1.445 -1.222 Td
(of Mixer )Tj
-1.14 -1.167 Td
(Performance )Tj
9.835 3.556 Td
(On-Column )Tj
-0.445 -1.167 Td
(Amplification )Tj
0.973 -1.222 Td
(of Mixing )Tj
0.666 -1.167 Td
(Ripple )Tj
9.847 3.556 Td
(Mixing )Tj
-2.028 -1.167 Td
(Ripple-Induced )Tj
0.804 -1.222 Td
(Peak Shape )Tj
0.446 -1.167 Td
(Distortion )Tj
/TT0 1 Tf
15.7562 0 0 15.7561 1655.735 1526.2159 Tm
(System )Tj
6.992 0.6 Td
(UltiMate)Tj
( 3000 Binary )Tj
3.307 -1.188 Td
(RSLC )Tj
7.38 1.188 Td
(UltiMate)Tj
( 3000 Binary )Tj
3.307 -1.188 Td
(RSLC )Tj
9.389 1.188 Td
(UltiMate)Tj
( 3000 )Tj
-0.889 -1.188 Td
(Quaternary RSLC )Tj
-0.055 Tw -29.487 -2.983 Td
[(Solvent A)55( )]TJ
0 Tw 10.318 0 Td
[(W)37(ater )]TJ
8.075 0.6 Td
[(W)37(ater/Acetonitrile )]TJ
0.059 -1.188 Td
[(99:1 + 0.1 % )18(TF)55(A)55( )]TJ
10.79 0.588 Td
[(W)37(ater + 0.12%TF)55(A)55( )]TJ
-29.242 -3.571 Td
(Solvent B )Tj
8.33 0.6 Td
[(W)37(ater + 0.07% )]TJ
1.468 -1.187 Td
(Acetone )Tj
8.631 1.187 Td
(Acetonitrile 100% )Tj
-0.018 Tw 1.274 -1.187 Td
[(+ )-18(0.1% TF)55(A)55( )]TJ
0 Tw 9.668 0.587 Td
[(ACN + 0.12% )18(TF)55(A)55( )]TJ
-29.371 -3.571 Td
(Gradient )Tj
7.52 0.6 Td
(Sinus-like gradient )Tj
-0.362 -1.187 Td
(between 10 \22670% B )Tj
11.329 0.588 Td
(5% B \(dial-a-mix\) )Tj
10.917 0.6 Td
(30\22640% for 6 min, )Tj
0.695 -1.187 Td
(100% for 1 min )Tj
-30.098 -3.223 Td
(Column )Tj
18.069 1.2 Td
(Acclaim)Tj
10.5041 0 0 10.5041 1995.5809 1321.0277 Tm
(\231)Tj
15.7562 0 0 15.7561 2006.0851 1316.3009 Tm
( 120 C18, )Tj
-2.76 -1.188 Td
(3 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(m, 120 )Tj
(\305)Tj
(, )Tj
-0.226 -1.187 Td
(250 )Tj
/C2_1 1 Tf
<0002>Tj
/TT0 1 Tf
( 3.0 mm )Tj
11.265 2.375 Td
(Phenomenex)Tj
( )Tj
-1.917 -1.188 Td
(SecurityGuard)Tj
10.5041 0 0 10.5041 2207.0359 1302.3167 Tm
(\231)Tj
15.7562 0 0 15.7561 2217.54 1297.5898 Tm
(, C18, )Tj
-4.849 -1.187 Td
( 4 )Tj
/C2_1 1 Tf
<0002>Tj
/TT0 1 Tf
( 3.0 mm )Tj
-30.807 -2.035 Td
[(T)111(emperature )]TJ
0 -1.187 TD
( )Tj
21.064 0.587 Td
(35 \260C )Tj
11.141 0 Td
(30 \260C )Tj
-32.205 -3.571 Td
(Flow Rate )Tj
8.882 0 Td
(1.00 mL/min )Tj
10.687 0 Td
(1.00 mL/min )Tj
11.141 0 Td
(1.00 mL/min )Tj
0.662 1 0.232 0.326 k
/TT1 1 Tf
29.5429 0 0 29.5427 1649.26 1075.4852 Tm
(Summary)Tj
( )Tj
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
17.7257 0 0 17.7256 1649.26 1043.9734 Tm
(An accurate way to experimentally determine the mixing performance using\
a )Tj
0 -1.167 TD
[(sinus-like gradient was described. )18(The correlation between signal attenuation )]TJ
0 -1.222 TD
(and mixer dwell volume follows an exponential decay curve. )Tj
0 -1.722 TD
[(It was shown that the amplifying ef)18(fect of the column on mixing ripples in case )]TJ
0 -1.222 TD
[(of )18(TF)55(A)55( as an ion-pairing agent is significant. With an experimentally-determin\
ed )]TJ
0 -1.222 TD
(amplification factor of up to 58, special attention to the mixer is need\
ed for best )Tj
0 -1.167 TD
(possible LOD. )Tj
0 -1.778 TD
(Mixing ripples can have diverse impacts on the liquid chromatographic )Tj
0 -1.167 TD
(performance, which does not necessarily provide conclusions based on its\
)Tj
0 -1.222 TD
(origin. It was shown that mixing ripples can significantly influence the\
peak )Tj
0 -1.222 TD
[(shape, although they aren\222t seen in the baseline. )18(The partial elution of )]TJ
0 -1.167 TD
(lysozyme can be seen as additional ripples on top of the peak. )Tj
0.662 1 0.232 0.326 k
/TT1 1 Tf
29.5429 0 0 29.5427 1649.26 739.6834 Tm
(References )Tj
0.75 0.68 0.655 0.902 k
/TT0 1 Tf
17.7257 0 0 17.7256 1649.26 708.1713 Tm
(1.)Tj
/C2_3 1 Tf
<0001>Tj
/TT0 1 Tf
1.5 0 Td
[(Dolan, J. W)55(.; Snyder)55(, L. R. Maintaining Fixed Band Spacing When )]TJ
0 -1.167 Td
(Changing Column Dimensions in Gradient Elution. )Tj
/TT3 1 Tf
22.567 0 Td
(J. )Tj
(Chromatogr)Tj
-0.037 Tc 6.298 0 Td
[(.)-37( A )]TJ
/TT1 1 Tf
0 Tc 1.426 0 Td
(1998)Tj
/TT0 1 Tf
(, )Tj
/TT3 1 Tf
-30.292 -1.222 Td
(799)Tj
/TT0 1 Tf
( \(1\2262\), 21\22634. )Tj
-1.5 -1.667 Td
(2.)Tj
1.222 0 Td
( )Tj
[(Winkler)55(, G. Increasing the Sensitivity of UV Detection in Protein and )]TJ
0.278 -1.167 Td
[(Peptide Separations When Using )18(TF)55(A)55( [)]TJ
17.325 0 Td
(trifluoroacetic)Tj
( acid]-Acetonitrile )Tj
-17.325 -1.222 Td
(Gradients)Tj
/TT3 1 Tf
(. LC-GC )Tj
/TT1 1 Tf
(1987)Tj
/TT0 1 Tf
( \(12\) 1044\2261045. )Tj
-1.5 -1.611 Td
(3.)Tj
1.222 0 Td
( )Tj
(Lamotte)Tj
(, S. )Tj
(Synthese)Tj
(, )Tj
(Charakterisierung)Tj
( und )Tj
20.456 0 Td
(Anwendung)Tj
( von )Tj
(station\344ren)Tj
( )Tj
-20.178 -1.222 Td
(Phasen)Tj
( auf der Basis von 1,5 )Tj
/C2_1 1 Tf
<0001>Tj
/TT0 1 Tf
(m )Tj
(unpor\366sem)Tj
( )Tj
(Kieselgel)Tj
( in der )Tj
(schnellen)Tj
( )Tj
0 -1.222 TD
[(HPLC, PhD )18(Thesis, Saarland University)74(, )]TJ
18.081 0 Td
(Saarbruecken)Tj
( 1998, 49.)Tj
0.662 1 0.232 0.326 k
10.674 0 Td
( )Tj
ET
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