Compact Ultrahigh-Pressure Nanoflow Capillary ... - ACS Publications

Article

Subscriber access provided by RYERSON UNIVERSITY

Compact Ultra-high Pressure Nano-flow Capillary Liquid Chromatograph Xiaofeng Zhao, Xiaofeng Xie, Sonika Sharma, Luke T Tolley, Alex Plistil, Hal E. Barnett, Martin Brisbin, Adam C. Swensen, John Calvin Price, Paul B Farnsworth, H. Dennis Tolley, Stanley D. Stearns, and Milton L. Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03575 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1

Compact Ultra-high Pressure Nano-flow Capillary Liquid Chromatograph

2 3

Xiaofeng Zhao,†a Xiaofeng Xie,†a Sonika Sharma,† Luke T. Tolley,‡ Alex Plistil,§ Hal E.

4

Barnett,§ Martin P. Brisbin,§ Adam C. Swensen,† John C. Price,† Paul B. Farnsworth,† H. Dennis

5

Tolley,|| Stanley D. Stearns,§ and Milton L. Lee*, †

6 7

†Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA

8 9

‡

Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL 62901, USA

10

§

11

||

12

†a

13

*

14 15

ABSTRACT: A compact ultra-high pressure nano-flow liquid chromatograph (LC) was

16

developed with the purpose in mind of creating a portable system that could be easily moved to

17

various testing locations or placed in close proximity to other instruments for optimal coupling,

18

such as with mass spectrometry (MS). The system utilized innovative nano-flow pumps,

19

integrated with very low volume stop-flow injector and mixing tee. The system weighed only 5.9

20

kg (13 lbs), or 4.5 kg (10 lbs) without controller, and could hold up to 1100 bar (16,000 psi)

21

pressure. The total volume pump capacity was 60 µL. In this study, the sample injection volume

22

was determined by either a 60 nL internal sample groove machined in a high pressure valve rotor

23

or by a 1 µL external sample loop, although other sample grooves or loops could be selected.

24

The gradient dwell volume was approximately 640 nL, which allowed significant reduction in

25

sample analysis time. Gradient performance was evaluated by determining the gradient step

26

accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient

27

reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon

VICI, Valco Instruments, Houston, Texas 77055, USA

Department of Statistics, Brigham Young University, Provo, UT 84602, USA These two authors contributed equally to this work.

Corresponding author: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28

mixture on a commercial 150 µm inner diameter capillary column packed with 1.7 µm particles.

29

Good retention time reproducibility (RSD < 0.17%) demonstrated that the pumping system could

30

successfully generate ultra-high pressures for use in capillary LC. The system was successfully

31

coupled to an LTQ Orbitrap MS in a simple and efficient way; LC-MS of a trypsin-digested

32

bovine serum albumin (BSA) sample provided narrow peaks, short dwell time and good peptide

33

coverage.

34 35 36

Introduction Ultra-high pressure liquid chromatography (UHPLC) has been around for almost two

37

decades, introduced to overcome the pressure limitations of conventional pumping systems and

38

to allow the use of very small particles as stationary phases.1-3 Due to its excellent separation

39

efficiency, short analysis time, low solvent consumption and easy coupling to mass spectrometry

40

(MS), UHPLC has become a very popular and green analytical tool, and is becoming widely

41

used for environmental, biochemical and pharmaceutical analysis.4-10 Very small particles (sub-2

42

µm) can result in improved performance in LC, as well as high speed separations.1,11-14 However,

43

without shortening the column length, the use of small particles requires high pressures. While

44

typically not an issue with small diameter capillary columns, operating at high pressures can

45

create frictional heating, which can impact the retention of solutes and, in some cases, cause

46

decomposition1,14 and loss in column efficiency.15,16 Heat dissipation can be facilitated by

47

reducing the column diameter.1,12,17 Small diameter packed fused silica columns allow fast heat

48

dissipation, low organic solvent usage and minimal waste generation.

49 50

The first UHPLC system for capillary columns packed with 1.5 µm nonporous silica particles was a home-built system reported by Jorgenson’s group.1 The same group also


Page 2 of 23

Page 3 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


51

constructed a gradient UHPLC system and separated a tryptic protein digest using 1.0 µm

52

particles at 9000 bar.2 However, the system was very large, inconvenient, and not amenable to

53

routine use. A subsequent UHPLC system was constructed by modifying a Waters Model 6000

54

pump, which was coupled to a tandem MS using a capillary packed with 1.5 µm nonporous

55

particles.18 This system demonstrated a high duty cycle, good signal-to-noise and full automation.

56

This work led to the introduction of commercial UHPLC instrumentation in 2004.19 Currently,

57

more than 20 different UHPLC systems are commercially available, with upper pressure limits

58

between 600-1500 bar.4,20,21 An excellent review by Eeltink et al.22 of advancements in UHPLC

59

instrumentation and practice were recently published. This review includes improvements in

60

column technology, pump design, sample introduction, temperature effects, and methods

61

development. UHPLC systems with pressures up to 20,000 psi, submicron porous particles and

62

automated switching valves with large external sample loops (5-10 µL) are readily available.

63

However, most of these systems are limited to laboratory bench operation, and cannot be easily

64

moved from one place to another or used for field applications. Furthermore, when coupled to

65

MS or other instruments in the laboratory, long connecting tubing is often needed, which can

66

greatly reduce the performance and throughput of the UHPLC system.

67

Compared to standard columns, e.g., 4.6-1.0 mm i.d., extra-column band broadening

68

becomes much more critical when using smaller internal diameter columns.21 Fekete et al.

69

compared the extra-column peak variance contributions of several commercially available LC

70

systems, and concluded that losses in efficiency can reach 30–55% with these UHPLC systems,

71

even when optimized.23 Further improvements in instrumental design could be achieved by using

72

smaller volume capillary fitting, smaller and shorter connecting capillary tubing and a smaller



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

73

volume detector cell to reduce detrimental extra-column dead-volume.21, 23-27 Furthermore, small

74

dwell volume is also highly desirable for UHPLC.21

75

The injection system for UHPLC should hold extremely high pressure, deliver a

76

reproducible small volume, eliminate all carryover and minimize brand spreading.28 Static-split

77

injection technology was reported by Jorgenson’s group, which withstood high pressures.1,2,29

78

Wu et al. used a pressure-balanced injection method to introduce sample, and found it to be

79

effective and reproducible.3 A commercial system (Waters ACQUITY UPLC) uses a flow-

80

through-needle injector.30 The gradient passes through the needle during injection, ensuring

81

complete sample transfer. In addition to the above-mentioned concerns, the detector data-

82

acquisition rate is also very important. Narrow peaks (as small as 1 s in some cases) require at

83

least 10-15 data points per peak to accurately specify resolution.21

84

Significant effort has been made in the past to miniaturize LC.31-35 However, little effort

85

has been made to miniaturize UHPLC. Recently, we reported a compact gradient LC system with

86

an LED UV-absorption detector (260 nm).32 This system meets many of the requirements for

87

field-portability, i.e., light-weight (9 lbs), low mobile phase consumption and waste generation,

88

and battery operation. The pressure generated by this pump reaches up to 8,000 psi. Although

89

nano-flow UHPLC systems are commercially available, they are not yet portable. With parallel

90

successful efforts in miniaturization of MS, compact UHPLC-MS can be expected in the future,

91

which would certainly be attractive for field analysis.36

92

An important application for UHPLC is MS-based proteomics.37 Due to the complex

93

nature of protein/peptide samples, integrated UHPLC-MS is commonly used to analyze these

94

samples. UHPLC is used prior to MS to separate complex mixtures of peptides or proteins for

95

accurate identification and quantitation. By using long columns, high pressures, small particles


Page 4 of 23

Page 5 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


96

and long gradients, peak capacities as large as 1500 have been achieved for UHPLC-MS based

97

proteomics.38 Iwasaki et al. identified 41,319 typtic peptides from 5,970 proteins using an 8 h

98

gradient and 4 m long monolithic column.39 High separation power, fast analysis, small sample

99

volume and easy coupling to MS are some of the key requirements for an UHPLC system used

100

for proteomics. As demonstrated in our previous research, one important aspect of miniature LC

101

is small dwell volume.32 For LC-MS based proteomics, small dwell volume will lead to less

102

extra-column band broadening, higher efficiency, lower carryover, faster analysis (i.e., less time

103

for sample to reach the head of the column), and higher sample throughput. In addition, a

104

compact LC will facilitate optimum positioning with the MS inlet. A miniature LC also allows

105

coupling of multiple LC units with a single MS to further improve sample throughput.40 A

106

simplified portable UHPLC system would reduce the footprint of the system, increase the duty

107

cycle, simplify maintenance, reduce costs and improve separation power by reducing extra-

108

column volume.

109

In this work, a small, portable gradient ultra-high pressure nano-flow pumping system

110

was developed and evaluated using an on-column LED-based UV-absorption detector reported

111

earlier, 41 as well as with an LTQ Orbitrap MS. The system was designed specifically for

112

capillary columns. Gradient performance and separation were evaluated using this new

113

integrated UHPLC-UV system. Efficient coupling of the nano-flow UHPLC to an Orbitrap MS

114

for analyzing trypsin-digested BSA for proteomics was demonstrated.

115

Experimental section

116

Chemicals and reagents

117 118

Water, methanol, propyl paraben, and three polycyclic aromatic hydrocarbon (PAH) standards (fluorene, anthracene, and benzo[a]pyrene) were purchased from Sigma-Aldrich (St.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

119

Louis, MO, USA). Acetonitrile (ACN) was purchased from Fisher Scientific (Pittsburgh, PA,

120

USA). All solvents and chemicals were HPLC or analytical grade. Chromatographically purified

121

bovine serum albumin (Sigma-Aldrich) ≥ 95% was prepared for bottom-up mass spectrometry

122

analysis using a filter-aided sample preparation (FASP) technique.42 Approximately 500 µg of

123

protein were loaded onto a 30 kDa cut-off ultra-filtration spin filter (Vivicon) for the FASP

124

protocol. Due to the high concentration of the final BSA peptide mixture, it was diluted 100 or

125

1000 fold and cleaned once more with a C-18 solid phase extraction cartridge (Sigma-Aldrich)

126

prior to injection into the LC.

127

Instrumentation

128

The pumping system for the new gradient nano-flow UHPLC instrument (Figure 1) was

129

engineered at VICI Valco Instruments (Houston, TX, USA). Compared to our previous gradient

130

pumping system, several important modifications were made as follows: (1) the “Y” design (i.e.,

131

two pumps and one valve) was changed to two individual pumps (each with stepper motor and

132

high-pressure valve), which were connected to a separate injection valve (Figure 1); (2) a new

133

proprietary sealing material was used to seal around the piston; and (3) smaller i.d. tubing was

134

used to reduce the dwell volume. The “Y” design used a size 11 actuator, which could only hold

135

up to 8,000 psi (550 bar) pressure, while the new design uses a size 17 actuator, which is more

136

accurate and can hold much higher pressure.

137

Overall, the pumping system included two pumps, each with its own high pressure valve

138

and stepper motor, a three-way static mixing tee connected to a serpentine tube for solvent

139

mixing, and another high pressure valve (18,000 psi) with a stop-flow injector. The pumping

140

system weighed 5.9 kg (13 lbs) and could hold up to 1100 bar (16,000 psi) pressure. Each pump

141

volume capacity was 30 µL. The stop-flow injector contained a sample groove in the rotor,with


Page 6 of 23

Page 7 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


142

sample volume of 60 nL, or a 1 µL external sample loop; however, the groove or external sample

143

loop could be engineered to deliver sample volumes anywhere between 5 nL and the 10 µL

144

range. Zero dead-volume (360 µm) fittings especially designed for capillary columns were used

145

for all connections, and no splitting was employed. This system provided accurate non-split flow

146

in the low nanoliter to microliter per minute range (as described previously33), and was fully

147

software-controlled. An on-column deep UV LED (260 nm) absorption detector described

148

earlier,41 or an LTQ Orbitrap MS (described later in this section) was used for detection.

149

System evaluation

150

The methods used to determine the dwell volume and gradient step accuracy were

151

reported earlier.31 In brief, methanol and propyl paraben in methanol were used as mobile phases

152

A and B, respectively. A linear gradient was programmed through a hollow capillary (150 µm

153

i.d.) from 0 to 100% B in 10 min with an isocratic hold at 100% B for several min. An injection

154

of uracil was used to determine the capillary dead volume. The dwell volume of the system was

155

calculated by subtracting the capillary dead volume from the gradient delay volume.

156

For evaluating step accuracy, the system was programmed from 0% B to 100% B 10%

157

intervals. The gradient was stopped every 10% step for several minutes to record the UV-

158

absorption signal. The absorbance recorded by the detector increased as the proportion of B in

159

the mobile phase increased, and reached a maximum at 100% B. Every measurement was

160

repeated at least four times to determine the precision of the results. The recorded absorbance

161

value at each step was divided by the absorbance recorded at 100% B, which served as a

162

reference.

163 164

Injection carry-over was studied by recording the signal obtained by first injecting uracil (0.33 mg/mL in an ACN/water mixture, 70:30 v/v). Then, the injector was thoroughly cleaned by



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

165

purging with mobile phase, and a blank injection (mobile phase) was made. Isocratic elution was

166

used (ACN/H2O, 70:30 v/v), and the flow rate was 2 µL/min. These experiments were repeated

167

three times, and the detector signals were recorded. For each experiment, the peak area in the

168

blank divided by the peak area of an analyte signal, multiplied by 100%, was reported as the

169

percent carry-over.

170

Chromatographic performance was evaluated using a packed capillary column

171

(ACQUITY BEH130 C18, 1.7 µm, 100 mm × 150 µm i.d., Waters, Milford, MA) to separate a

172

PAH mixture under gradient programming conditions. The post-column capillary was removed

173

and replaced by a UV-transparent fused silica capillary (150 µm i.d.), which was used as a

174

detection window. Gradient elution was programmed from 50% A to 100% B in 11.6 min.

175

Solvent A was water and solvent B was ACN. The flow rate was 2 µL/min. The retention time

176

reproducibilities (n = 4) of components were calculated.

177

Coupling to an LTQ Orbitrap MS

178

Polyimide coated fused silica tubing (75 µm i.d. × 365 µm o.d.) was used to prepare a

179

column for coupling to the MS. Before packing, one end of the capillary was melted using a

180

small butane micro torch, and pulled into a narrow tip (approximate 25 µm i.d.) to be used for

181

electrospray ionization (ESI). Then, the capillary was packed with 1.7 µm BEH C18 particles

182

(Waters, Milford, MA) by slurry packing. After the desired length (approximately 10 cm) was

183

packed, the column was flushed with ACN before being used for MS analysis.

184

A hollow capillary (25 µm i.d. × 365 µm o.d., 15 cm total) was used for the transfer line

185

between the column with integrated ESI tip and injection valve. A VICI zero dead volume union

186

was used to connect the transfer line and the column as well as to apply the high voltage for

187

electrospray (photographs of all connections and column with integrated electrospray tip are


Page 8 of 23

Page 9 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


188

shown in the Supplemental Information, Figures S1-S2). MS detection was carried out after ESI

189

in the positive-ion full-scan mode. An LTQ Velos Pro-Orbitrap Elite tandem MS from Thermal

190

Fisher (Waltham, MA USA) was used, and the settings of the MS were as follows: spray voltage,

191

2.5 kV; capillary temperature, 325oC; and scan range, 150-2000 m/z. Nitrogen was used as

192

sheath gas (3 arb. units). Data acquisition and analysis were performed using Thermo Xcalibur

193

software Version 2.2.

194

Results and discussion

195

Pump operation

196

The UHPLC system contained a four-port injection valve to switch between sample

197

loading and injection (Figure 2). Each port served a specific purpose as described in the

198

following: S is the sample injection port, W is the waste port, C is the column port, and M is the

199

port through which the mixed mobile phase flowed to the internal sample groove and then to the

200

column and detector. SG represents the sample groove in the rotor for loading the sample.

201

Software controls the pump to switch automatically between the “load” and “inject”

202

positions. In the load position, the pumps were automatically refilled with their respective mobile

203

phases A and B at the preset rate of 20 µL/min through a 200 µm i.d. tube. At the same time, the

204

sample was introduced manually into the 60 nL sample loop. After the valve was switched to the

205

inject position (i.e., the rotor rotated by 90o), solvents from the pumps were mixed at the tee to

206

form a gradient flow. Then this gradient flow was used to carry the sample in the sample loop

207

through the analytical column to the detector for analysis.

208

Dwell volume

209 210

The dwell volume is defined as the volume from the solvent mixing point to the head of the analytical column.43 The sample experiences undesirable isocratic broadening in the dwell



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

211

volume. When performing fast separations in LC, there are two main concerns related to large

212

system dwell volume: (1) unreliable gradient method transfer between columns of different

213

geometries, and (2) greater than expected separation time.21 Small dwell volume is highly

214

recommended for fast analysis, especially when small i.d. columns are used, such as capillary

215

columns. Knowing the dwell volume of a new system is also very critical for transferring

216

methods from conventional LC to UHPLC.

217

The dwell volume was calculated by subtracting the capillary dead volume from the

218

gradient delay volume. The dead volume was 1.91 µL (1.91 min × 1.0 µL/min), so the dwell

219

volume of this system was calculated to be 0.64 ± 0.008 µL. This value is less than half of the

220

dwell volume measured for our previous nano-flow gradient system (1.3 µL),32 and less than the

221

delay volume of a Waters NanoACQUITY UPLC system (< 1 µL).

222

Gradient profile

223

Page 10 of 23

Good gradient accuracy and precision is a prerequisite for achieving reproducible

224

separations. To test the accuracy of the gradient, experimental step errors were plotted against

225

their corresponding theoretical step percentage values as illustrated in Figure 3A. Deviations

226

from predicted values were less than 0.8%. A gradient step accuracy of ± 1% is considered to be

227

acceptable for an LC system.32 In order to evaluate the precision of the gradient, experimental

228

RSD values were calculated for a series of step percentages and plotted against their respective

229

theoretical step percentages. As shown in Figure 3B, the RSD values (n = 4) were less than 0.6%,

230

which is comparable to commercial instruments. Therefore, the gradient performance of this

231

system was found to be excellent in terms of step accuracy and precision.

232 233

Low carry-over is important for quantitative analysis in LC. Uracil was chosen as test analyte because a non-retained marker is commonly used to evaluate the injector performance.


Page 11 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


234

Data from this work showed that the nano-flow pump injector had low carry-over (0.20 ± 0.1%).

235

This indicated that the flow-through injector is very reliable for quantitative work.

236

Reproducible retention time is a fundamental requirement for chromatographic

237

separations. Under gradient conditions, high reproducibility comes from high gradient accuracy.

238

As shown in Figure 4, highly reproducible retention times of PAHs were obtained by using the

239

UHPLC system. The RSD values were calculated to be less than 0.12%, which is better than the

240

value (1.42%) obtained from our previous “Y” design gradient pumping system.32 Day-to-day

241

reproducibility was also measured from experiments conducted over three days with three

242

injections per day. The RSD was less than 0.17% for these PAH compounds. It should be noted

243

that there was still some dead-volume between the column and UV detector, so separation

244

performance could be further improved if this dead-volume was removed.

245

Performance under high pressure and coupling with MS

246

Another nano-flow UHPLC system with 1.0 µL sample loop was coupled to an LTQ

247

Orbitrap MS for proteomic analysis. Due to the small particle size (1.7 µm) used in the analysis,

248

higher backpressure (>10,000 psi) was required. Under high pressure, solvents compress to a

249

certain extent, which leads to inaccurate flow rate and a distorted gradient. Most commercial

250

pumping systems use reciprocating designs, while some use flow and pressure feedback control

251

to deal with solvent compressibility. In our system, for simplicity, no flow and pressure sensors

252

were employed.

253

Therefore, in order to obtain the desired linear gradient, we evaluated two different ways

254

to deal with solvent compressibility. In the first approach, at the beginning, both pumps were

255

moved at the same speed until pressure was built up, and then the pump for solvent A (water)

256

was programmed to pump at full speed while the other pump was temporarily stopped to wash



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 23

257

all of solvent B (acetonitrile) out of the line. Sample was then injected into the flow stream and

258

the elution gradient was started. Figure 3S in the Supplemental Information shows the gradient

259

profile for operating the pumping system in this manner. After the point of sample injection, a

260

near-linear gradient was generated. For the second approach, a series of gradient steps was

261

generated at the first of the run to compensate for the solvent compressibility. As shown in

262

Figure 5A, without an initial compensation procedure, the gradient was not linear, but jumped

263

rapidly from 0 to 40% solvent B (uracil was added to solvent B for easy detection) with a steep

264

slope at the beginning. By adding two gradient steps (gradient solvent B at 25% for 10 min and

265

then 5% for 10 min), a near linear gradient from 5% to 40% was obtained as shown in Figure 5A.

266

Both gradient compensation approaches were used to separate a trypsin-digested bovine

267

serum albumin (BSA) sample. Using the method of delay injection, a 25 min gradient from 5%

268

to 45% acetonitrile was used, for the chromatogram shown in Figure 5B. The FWHM peak width

269

was approximately 10 s, the dwell volume was less than 2 µL, and an identified peptide coverage

270

of over 90% was obtained using Mascot.44 Similar results were obtained using stepwise gradient

271

compensation as shown in Figure 5C, with a gradient from 5% to 30% in 30 min and FWHM of

272

approximately 15 s. Compared to commercial nano-flow LC systems that exhibit dwell times of

273

over 10 min (> 4 µL in volume), peptide coverages of 67% to 87%, and peak widths (FWHM) of

274

approximately 20 s (3-5 µm particles),45 the dwell time in our system was less than 4 min (using

275

delayed injection), which improved the duty cycle better than 20%. Further work is currently

276

being done to implement an initial solvent compression routine into the operating procedure.

277

Conclusions

278 279

Ultra-high operating pressures for capillary LC were made possible using a newly developed LC pumping system. This new nano-flow gradient system gave highly reproducible


Page 13 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


280

retention times of PAH compounds on a capillary column packed with sub-2 µm particles.

281

Efficient gradient separation of peptides was also demonstrated when coupled to an LTQ

282

Orbitrap MS. The features of the capillary nano-flow UHPLC system described in this study

283

include: (1) it is simple to operate and maintain, (2) it consumes a minimum amount of solvent,

284

(3) it is easy to position close to the electrospray source (which greatly reduces band broadening

285

and analysis time, and improves detection limits), and (4) it provides high resolution LC of

286

complex peptide mixtures for proteomics studies. Two simple approaches were demonstrated to

287

address solvent compressibility during initial pressurization of the system for ultrahigh pressure

288

operation.

289

Supporting Information

290

S1. Photographs of a column with integrated electrospray tip.

291

S2. Photographs showing the interface between the nano-flow UHPLC system and

292 293

Orbitrap MS. S3. Gradient profile obtained when both gradient pumps were initially moved at the same

294

speed until pressure was built up, and then the pump for solvent A was programmed to pump at

295

full speed while the other pump was temporarily stopped to wash solvent B out of the line.

296 297 298

REFERENCES

299

(1) MacNair, J. E.; Lewis, K. C.; Jorgenson, J. W. Anal. Chem. 1997, 69, 983-989.

300

(2) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700-708.

301

(3) Wu, N.; Lippert, J. A.; Lee, M. L. J. Chromatogr. A 2001, 911, 1-12.

302

(4) De Vos, J.; Broeckhoven, K.; Eeltink, S. Anal. Chem. 2016, 88, 262-278.

303

(5) Bouvier, E. S. P.; Koza, S. M. TrAC, Trends Anal. Chem. 2014, 63, 85-94. 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

304 305

(6) Gumustas, M.; Kurbanoglu, S.; Uslu, B.; Ozkan, S. A. Chromatographia 2013, 76, 13651427.

306

(7) Denoroy, L.; Zimmer, L.; Renaud, B.; Parrot, S. J. Chromatogr. B 2013, 927, 37-53.

307

(8) Purcaro, G.; Moret, S.; Bučar-Miklavčič, M.; Conte, L. S. J. Sep. Sci. 2012, 35, 922-928.

308

(9) Kumar, A.; Saini, G.; Nair, A.; Sharma, R. Acta Pol. Pharm. 2012, 69, 371-380.

309

(10) Howard, J. W.; Kay, R. G.; Pleasance, S.; Creaser, C. S. Bioanalysis 2012, 4, 2971-2988.

310

(11) Broeckhoven, K.; Desmet, G. TrAC, Trends Anal. Chem. 2014, 63, 65-75.

311

(12) Jorgenson, J. W. Annu. Rev. Anal. Chem. 2010, 3, 129-150.

312

(13) Anspach, J. A.; Maloney, T. D.; Colón, L. A. J. Sep. Sci. 2007, 30, 1207-1213.

313

(14) Nováková, L.; Matysová, L.; Solich, P. Talanta 2006, 68, 908-918.

314

(15) Gritti, F.; Martin, M.; Guiochon, G. Anal. Chem. 2009, 81, 3365-3384.

315

(16) Gritti, F.; Guiochon, G. J. Chromatogr. A 2007, 1138, 141-157.

316

(17) Colón, L. A.; Cintrón, J. M.; Anspach, J. A.; Fermier, A. M.; Swinney, K. A. Analyst 2004,

317

129, 503-504.

318

(18) Tolley, L.; Jorgenson, J. W.; Moseley, M. A. Anal. Chem. 2001, 73, 2985-2991.

319

(19) Mazzeo, J. R.; D. Neue, U.; Kele, M.; Plumb, R. S. Anal. Chem. 2005, 77, 460 A-467 A.

320

(20) Fekete, S.; Schappler, J.; Veuthey, J. L.; Guillarme, D. TrAC, Trends Anal. Chem. 2014, 63,

321

2-13.

322

(21) Fekete, S.; Kohler, I.; Rudaz, S.; Guillarme, D. J. Pharm. Biomed. Anal. 2014, 87, 105-119.

323

(22) De Vos, J.; Broeckhoven, K.; Eeltink, S. Anal. Chem. 2016, 88, 262-278.

324

(23) Fekete, S.; Fekete, J. J. Chromatogr. A 2011, 1218, 5286-5291.

325

(24) Wu, N.; Bradley, A. C.; Welch, C. J.; Zhang, L. J. Sep. Sci. 2012, 35, 2018-2025.


Page 14 of 23

Page 15 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

326 327 328 329 330 331


(25) Omamogho, J. O.; Hanrahan, J. P.; Tobin, J.; Glennon, J. D. J. Chromatogr. A 2011, 1218, 1942-1953. (26) Alexander, A. J.; Waeghe, T. J.; Himes, K. W.; Tomasella, F. P.; Hooker, T. F. J. Chromatogr. A 2011, 1218, 5456-5469. (27) Gritti, F.; Sanchez, C. A.; Farkas, T.; Guiochon, G. J. Chromatogr. A 2010, 1217, 30003012.

332

(28) Swartz, M. E. J. Liq. Chrom. Relat. Tech. 2005, 28, 1253-1263.

333

(29) Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. A 1983, 255, 335-348.

334

(30) Waters product literature.

335

(31) Baram, G. I. J. Chromatogr. A 1996, 728, 387-399.

336

(32) Sharma, S.; Plistil, A.; Barnett, H. E.; Tolley, H. D.; Farnsworth, P. B.; Stearns, S. D.; Lee,

337 338 339 340 341

M. L. Anal. Chem. 2015, 87, 10457-10461. (33) Sharma, S.; Plistil, A.; Simpson, R. S.; Liu, K.; Farnsworth, P. B.; Stearns, S. D.; Lee, M. L. J. Chromatogr. A 2014, 1327, 80-89. (34) Sharma, S.; Tolley, L. T.; Tolley, H. D.; Plistil, A.; Stearns, S. D.; Lee, M. L. J. Chromatogr. A 2015, 1421, 38-47.

342

(35) Zhou, L.; Lu, J. J.; Gu, C.; Liu, S. Anal. Chem. 2014, 86, 12214-12219.

343

(36) Snyder, D. T.; Pulliam, C. J.; Ouyang, Z.; Cooks, R. G. Anal. Chem. 2016, 88, 2-29.

344

(37) Aebersold, R.; Mann, M. Nature 2003, 422 (6928), 198-207.(38) Shen, Y. F.; Zhao, R.;

345

Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K. Q.; Pasa-Tolic, L.; Veenstra, T. D.;

346

Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73 (8), 1766-1775.

347 348

(39) Iwasaki, M.; Sugiyama, N.; Tanaka, N.; Ishihama, Y. J. Chromatogr. A 2012, 1228, 292297.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

349

(40) Li, S.; Hao, Q.; Gounarides, J.; Wang, Y. K. J. Mass Spectrom. 2012, 47, 1074-1082.

350

(41) Sharma, S.; Tolley, H. D.; Farnsworth, P. B.; Lee, M. L. Anal. Chem. 2015, 87, 1381-1386.

351

(42) Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M., Nature methods 2009, 6 (5), 359-

352

62

353

(43) Li, Y.; Tolley, H. D.; Lee, M. L. J. Chromatogr. A 2011, 1218,1399−1408.

354

(44) Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20,

355 356 357

3551-3567. (45) Liu, Q.; Cobb, J. S.; Johnson, J. L.; Wang, Q.; Agar, J. N. J. Chromatogr. Sci. 2014, 52, 120-127.

358 359 360 361 362


Page 16 of 23

Page 17 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


363

Figure captions

364

Figure 1. (A) Cut-away drawing of a single nano-flow pump; (B) photograph of the nano-flow

365

UHPLC with LED detector.

366

Figure 2. Operating schematics of the nano-flow UHPLC injection valve. (A) Load position, and

367

(B) inject position.

368

Figure 3. (A) Gradient step accuracy (±% solvent B) and (B) precision in gradient step accuracy.

369

Figure 4. Reproducibility in retention times of of PAHs.

370

Figure 5. (A) Comparison of gradient profiles (uracil was added to monitor the concentration of

371

solvent B, acetonitrile); blue: no compensation step with pump speed set to generate a linear

372

gradient from 0% to 40%; red: two step manual compensation, 10 min at 25% B and then 10 min

373

at 5% B, was used prior to starting the sample elution gradient. (B and C) Chromatograms of a

374

trypsin-digested BSA sample using coupled UHPLC-Orbitrap MS: (B) sample injected 20 min

375

after 10 min 50:50 (A/B, water/ACN) and 10 min 95:5 wash (total ion and extracted-ion

376

chromatograms, m/z = 632. (C) Sample injected at 0 min, compensation steps of 75:25

377

(water/CAN) for 10 min and 95:5 for 10 min before linear gradient of 5% to 30% was started

378

(total-ion and extracted-ion chromatograms, m/z = 654. Experimental conditions: column with

379

integrated ESI tip, 12 cm (75 µm i.d. × 365 µm o.d.); mobile phase A, 0.1% formic acid in

380

water/ACN (95:5, v/v); mobile phase B, 0.1% formic acid in ACN; flow rate at 500 nL/min;

381

injection volume, 1.0 µL.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

382 383

Figure 1


Page 18 of 23

Page 19 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


384 385

Figure 2

386



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408

Figure 3


Page 20 of 23

Page 21 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


409 410

Figure 4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

411

B

412

C

413 414

Figure 5 22 ACS Paragon Plus Environment

Page 22 of 23

Page 23 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

415


Abstract Graphic

416

417 418


Compact Ultrahigh-Pressure Nanoflow Capillary ... - ACS Publications

Recommend Documents