ApplicationNews

No.L445

High Performance Liquid Chromatography

Effective Method Development Using Online Blending of Ion-Pair Reagents for Mobile Phase Preparation

LAAN-A-LC-E221

Column

Auto injector

Detector

0.1% TFAaqPump A

Pump B

LPGEunit

LPGE unit

Gradient mixer

25

Ex.) Mixing Ratio Concentration of Ion-pair Regent 0.025 %

0

0

75

Water

Acetonitrile

Tetrahydrofuran

Methanol

Water

H3PO4aq

NaH2PO4aq

n Online Mobile Phase Blending System

n Effects on Retention Due to Concentration of Ion-Pair Reagent

When investigating analytical conditions during HPLC method development, selection of the mobile phase is extremely important due to its effect on analyte separation. In ion-pair reversed-phase mode analysis, the column retention strength changes depending on the concentration of the ion-pair reagent that is added. Therefore, the ion-pair reagent concentration is very important when considering the analytical conditions. However, f rom the ana lys t ' s perspect i ve , the preparation of several mobile phases with different concentrations of ion-pair reagent for each analysis in the investigation of the analytical conditions represents a tedious and time-consuming effort.Here, we introduce a technique in which multiple var iants of mobi le phase conta in ing d i fferent concentrations of ion-pair reagent are prepared online for use in analysis.

The ion-pair reagent concentration in the mobile phase was adjusted by mixing multiple solutions online using a pump equipped with a low-pressure gradient valve. Fig. 1 shows an example of the flow line diagram.This is a high-pressure gradient elution system in which each pump contains a low-pressure gradient valve that permits mixing of up to four different solutions in any desired ratio. This example illustrates the process in which aqueous mobile phase containing ion-pair reagent delivered by pump A and organic mobile phase*1 delivered by pump B are mixed entirely online.*1 For online preparation of organic mobile phase and aqueous

mobile phase, refer to Application News No. L443 and L444.

The ionic dissociation equilibrium state varies according to the pH of the solution in which an ionic compound is d i spersed. I f the ana ly te i s d i s soc ia ted , then hydrophilicity is increased, and, in reversed-phase mode, retention decreases. Thus, when the reversed-phase mode is used for separating these substances, it is common to use a mobile phase containing a buffer solution in a pH range where the analyte will not dissociate. However, when the target analyte is either st rongly ac id ic or st rongly bas ic , i t cannot be maintained in a non-dissociated state in a mobile phase that can be readily used for analysis because the extremes of pH required are not suitable for the column or the HPLC components. Also, even if the analyte is weakly acidic or weakly basic, analysis sometimes must be conducted using a mobile phase pH range that causes the analyte to dissociate, depending on the type of column used or the sample solubility. In such a case, reversed-phase ion-pair mode is applicable.It is possible to increase the retention in reversed-phase mode by adding an ionic substance having a charge opposi te to that of the analyte that has been dissociated in the mobile phase in order to form anion pair, thereby permitting separation of the target analyte. Generally, the degree of retention varies greatly depending on the type of ion-pair reagent, i ts concentration, and mobile phase pH*2. Therefore, careful consideration of these parameters is extremely important when setting up the analytical conditions. *2 The pH of the aqueous mobile phase can also be adjusted online.

This system permits mixing of up to four different solutions to allow online modification and adjustment of both the mobile phase pH and ion pair reagent concentrat ion. For more information about online pH adjustment of the aqueous mobile phase, please refer to ANL443.

Fig. 1 Flow Diagram of Online Mobile Phase Blending System

For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2013www.shimadzu.com/an/

ApplicationNews

No.L445

First Edition: Feb, 2013

No.Mixing ratio

(Ⅰ)/(Ⅱ)TFA Concentration

(%)(1) 10/90 0.01(2) 25/75 0.025(3) 50/50 0.05(4) 75/25 0.075(5) 100/0 0.1

n Online Blending of Ion-Pair ReagentFig. 2 shows the chromatograms obtained from analysis of a standard solution of Angiotensin Ⅱ. The mobile phases used were prepared online using various mixing ratios of (Ⅰ) 0.1 % trifluoroacetic acid (TFA) and (Ⅱ)water. The analytical conditions are shown in Tables 2 and 3.It is obvious that the retention time of Angiotensin Ⅱvaries greatly due to the concentration of TFA (serving as the ion-pair reagent in this analysis) in the mobile phase.

n Comparison of Chromatograms and Retention Time Repeatability Obtained Using Online- and Offline-Blended Mobile Phases

Fig. 3 shows the chromatograms obtained from five repeat analyses of AngiotensinⅡ using a 0.05 % TFA concentration in the mobile phase prepared using online mixing, and the chromatogram obtained using the same mobile phase mixed offline.The chromatograms obtained using online and offline mobile phase preparation show excellent correlation.In addition, the retention time repeatability obtained from five repeat analyses of the sample using online- and offline-blended mobile phases are shown in Table 4. The results obtained indicate excellent equivalence of retention time repeatability between the two methods.

0.0 1.0 2.0 3.0

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

275000

300000uV

1

TFA Conc.

(1) 0.01 %

(2) 0.025 %

(3) 0.05 %

(4) 0.075 %

(5) 0.1 %

Peak1: AngiotensinⅡ

min

Fig. 2 Chromatograms of Standard Solution of Angiotensin Ⅱ (1 mg/mL)

0.0 1.0 2.0 3.0

0

50000

100000

150000

200000

250000

300000

350000

400000

450000uV

1

Online blending

Offline blending

Peak1: AngiotensinⅡ

min

Fig. 3 Chromatograms Obtained Using Online Blending and Offline Blending of 0.05 % TFA (Ⅰ)/(Ⅱ) = 50/50

Column : Shim-pack XR-ODS (50 mm L. × 3.0 mm I.D., 2.2 μm)Mobile Phase : A: Online blended 0.01 ～ 0.1 % Trifluoroacetic acid（TFA） (Ⅰ) : 0.1 % TFA (Ⅱ) : Water Mixing Ratios of (Ⅰ)/(Ⅱ) are shown in Table 2 B: Acetonitrile A/B=8/2(v / v)Flowrate : 1.0 mL/minColumn Temp. : 40 °CDetection : SPD-M20A at (220 nm)Injection Vol. : 1 μL

Table 1 Analytical Conditions

CompoundRepeatability of retention time RSD (%) n5Online blending Offline blending

Angiotensin Ⅱ 0.191 0.24

Table 3 Repeatability of Retention Time

Table 2 Online Mixing Ratios of Solutions (Ⅰ) and (Ⅱ)