Electronic Supplementary Material for

Utilization of deep eutectic solvents as novel mobile phase additives for

improving the separation of bioactive quaternary alkaloidsTing Tana,b, Mingliang Zhangc, Yiqun Wana,b* and Hongdeng Qiuc,*

a State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, Chinab School of chemistry, Nanchang University, Nanchang 330031, China

c Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province,

Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China

*Corresponding author. Tel.:+86 931 4968877; fax: +86 931 8277088; E-mail: hdqiu@licp.cas.cn (H. Qiu). wanyiqun@ncu.edu.cn

(Y. Wan)

Fig. S1. The picture of DESs used in this work.

Characterization

Chemical composition was characterized by Fourier Transform Infrared Spectroscopy (FTIR)

(IFS 66v/s) (Bruker, Germany). The peaks in the fingerprint region (400-1300 cm -1) indicate that

hydrogen bonds were formed [49]. DESs displayed the characteristic peaks of –OH between 3000

cm-1 and 3500 cm-1.

Differential Scanning Calorimetry (DSC) was carried out by a DSC 822e (Mettler Toledo,

Switzerland) equipped with a Brookfield DV-III Ultra programmable rheometer and the value was

listed in Table 1.

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Fig. S2. Characterization of DES-1

Fig. S3. Effect of DESs in acetonitrile (a) and methanol (b), containing (a) 32 : 68 (v/v) MeCN : 1%

DES-1 (pH 3.3) and (b) 32 : 68 (v/v) MeOH : 1% DES-1 (pH 3.3). Peaks: 1, CopC; 2, Sang; 3,

BerbC and 4, Chel. Flow-rate: 1.0 mL min-1. Detection: UV 345 nm. Column: C18. Temperature: 30

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°C. Injection volume: 10 μL.

Preparation of 1-butyl-3-methylimidazolium chloride ionic liquid.

1-Methylimidazolium (8.21 g) and 1-chlorobutane (10.20 g) were mixed in a round-bottom

flask under N2. The reaction mixture was stirred at 70 °C for 72 h.

With comparison purposes, the separation result of DESs as mobile phase additives for

quaternary alkaloids was compared with commonly used IL. When adding 0.25% IL to water as

mobile phase additive, baseline separation (Fig. S4b) was obtained, and the retentions of four

analytes were slightly shorter than using DES-1.

Fig. S4. Separation of four QAs including CopC (1), Sang (2), BerbC (3) and Chel (4) with mobile

phase 32 : 68 (v/v) for (a) MeCN : 0.25% DES-1 (pH 3.3) and (b) MeCN : 0.25% 1-butyl-3-

methylimidazolium chloride aqueous solution (pH 3.3).

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Fig. S5. Effect of temperature on the retention time of analytes in C18 column.

Table S1. Analytical performance of QAs (n=3).

Quanterary

alkaloids

Linear range

(μg mL-1)

Linear equation r LOD

(μg mL-1)

LOQ

(μg mL-1)

Precision

RSD (%)

Repeatability

RSD (%)

CopC 0.10-25.0 Y=17177x+11421 0.9988 0.008 0.030 2.03 2.38

Sang 0.10-25.0 Y=19623x+6247.6 0.9992 0.020 0.060 0.95 1.86

BerbC 0.10-25.0 Y=32773x+14073 0.9992 0.006 0.020 0.37 1.01

Chel 0.10-25.0 Y=18258x+6407.1 0.9994 0.010 0.050 1.81 2.99

Notes: Y, peak area; x, concentration of QAs; r, correlation coefficient; LOD, limit of detection; LOQ, limit of

quantitation.

Table S2. Recoveries of BerbC in Lanqin Chinese herbal oral solution and BerbC tablet.

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Sample Found

(μg mL-1)

Spiked

(μg mL-1)

Detect

(μg mL-1)

Recovery

(%)

RSD

(%)

Lanqin Chinese herbal solutiona 5.75

1 6.69 99.1 2.64

5 9.86 91.7 1.70

7 11.8 92.5 2.85

BerbC tabletb 0.56

0.25 0.74 91.4 1.89

0.50 0.96 90.6 0.60

1.5 2.02 98.1 2.16

Notes: a, 10 fold diluted Lanqin Chinese herbal solution; b, one tablet was dissolved in 50 L deioned water.

Effect of DESs on the column stability

At the very beginning of the experiment, 80 : 20 (v/v) MeOH : H2O was used to balance the C18

column for 4 hours, four reference reagents including uracil, toluene, benzene and naphthalene were

injected 10 μL into HPLC-UV, under the detection conditions of flow-rate 1.0 mL min -1, 30 °C, 254

nm. The C18 column was used throughout the whole optimization of alkaloids separation and sam-

ple analysis. After experiment, for comparison purposes, four reference reagents were detected

again under the same condition. Identical experiments were carried out on C8.

Table S3. Performance of C18 and C8 column stability.

C18 C8

Before experiment After experiment Before experiment After experiment

RT(min) N (/m) P(Mpa) RT(min) N (/m) P(Mpa) RT(min

)

N (/m) P(Mpa) RT(min) N (/m) P(Mpa)

Uracil 1.75 11728 7.6 1.76 11945 7.6 1.80 15881 5.6 1.80 15806 5.6

Toluene 3.22 28189 3.23 28351 3.32 45200 3.32 45295

Benzene 4.11 31096 4.12 31204 4.07 53648 4.07 53843

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Naphthalene 4.92 34150 4.93 34230 4.48 59088 4.48 59185

Note: N, theoretical plate number; P, back pressure.

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