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Journal of Chromatography A, 1141 (2007) 182–190

An automated mobile phase preparation workstation

Kelly Swinney a,∗, Benjamin Young b, Matthew E. Jakubik b,Hinton Clark b, John Troisi b, Adam M. Fermier b

a Johnson & Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgiumb Johnson & Johnson Pharmaceutical Research and Development, 1000 Route 202, P.O. Box 300, Raritan, NJ 08869, USA

Received 11 November 2004; received in revised form 29 November 2006; accepted 1 December 2006Available online 16 December 2006

bstract

An automated solvent dispensing workstation capable of delivering volumes ranging from 10 mL to 4.5 L for the preparation of solutions/mobilehases was developed and implemented into the industrial R&D laboratory. The workstation was designed to address business, safety, and

ompliance needs while meeting or exceeding the precision and accuracy of current manual methods of preparation. The system’s performanceas optimized with respect to liquid transfer tubing inner diameter, pumping pressure, flow characteristics of the valve, and computer control logic.he automated solvent dispensing workstation was shown to exceed the specifications set by the ASTM for Class A graduated cylinders for allispense volumes (10 mL–4.5 L).

2006 Elsevier B.V. All rights reserved.

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eywords: Mobile phase preparation; Automation; Liquid chromatography; HP

. Introduction

In any laboratory setting, maintaining a safe and productiveork environment is essential. One approach to ensure that both

afety and productivity coexist is through the implementation ofutomation for tasks that are both repetitive and routine [2]. Oneuch task that has been identified as time consuming, routine, andmenable to automation is the preparation of mobile phases forigh performance liquid chromatography (HPLC). For example,n the pharmaceutical industry, HPLC is relied upon not only dur-ng manufacturing for quality analysis/quality control (QA/QC)ut also in R&D during the drug development process. It is notnusual for QA/QC laboratories to require the preparation of0s of liters of a variety of mobile phases daily. While the vol-me and variety of mobile phases required in R&D laboratoriesay vary each day, the volume that must be prepared is substan-

ial. Therefore to increase productivity and more importantly,ake better use of the highly trained scientists’ time, a system

apable of preparing HPLC mobile phases/mixed solutions orispensing solvents accurately and precisely is desirable.

∗ Corresponding author. Tel.: +32 14 60 5001; fax: +32 14 60 7083.E-mail address: kswinne1@prdbe.jnj.com (K. Swinney).

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Solution preparation

In addition to increase laboratory productivity, the implemen-ation of an automated mobile phase preparation workstationnto the laboratory would also provide a safer work environ-

ent. First and foremost, the automated system would decreasehe interaction of the scientist with potentially hazardous chemi-als. Second, automating the preparation of mobile phases wouldlso reduce the risk of chemical spills in the laboratory. Third,ecessary and appropriate labeling and notebook documentationf all prepared mobile phases could be automatically organizednto a standard form and printed by the system ensuring regula-ory compliance and eliminating one of the many documentationequirements of the scientist in a GLP/GMP work environment.ourth, a centralized system capable of servicing multiple labsr up to 50 scientists would help to drastically reduce the volumef bulk solvent being stored in the laboratories for mobile phasereparation helping to ensure that fire codes are being met atll times. Currently no commercial system exists that takes intoccount all of the above stated safety, regulatory and businesseeds [1].

Realizing the need for an automated mobile phase prepara-ion workstation capable of meeting or exceeding the accuracy,

recision, and preparation time of manually prepared mobilehases, a pressure-driven mass-based solvent delivery systemas designed and implemented. Presented here is a detailed dis-

ussion of the design and development of the automated mobile

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hase preparation workstation. A comprehensive description ofhe workstation’s computer control logic and user interface islso included. The effects of pressure, dispensing tube inneriameter, valve flow coefficient (CV), and solvent viscosity andurface tension on solvent delivery accuracy and precision willlso be covered. Finally, an optimized system capable of dispens-ng volumes ranging from 10 mL to 4.5 L for the preparation of

obile phases will be presented.

. Experimental

.1. A detailed description of the automated mobile phasereparation workstation

Shown in Fig. 1 is a picture of the automated mobile phasereparation workstation capable of preparing mobile phasesor HPLC. The workstation was designed about a 1 m × 1 mootprint that could be installed in a standard explosion proofaboratory ventilation hood. The system consisted of a series oftwo-way, normally closed (NC) solenoid valves (1-A) mounted

ide by side on a custom valve manifold (1-B) (Biafore Cad Con-epts, Pipersville, PA) that was bolted via a 90◦ angle bracketNT55-379, Edmund Optics, Barrington, NJ) to a 0.6 m × 0.3 mase plate (1-C) (NT03-640, Edmund Optics, Barrington, NJ).he 0.6 m × 0.3 m ft base plate was mounted vertically using two

ight angle brackets (1-D) (NT55-479, Edmund Optics, Barring-on, NJ) to a second 0.6 m × 0.3 m base plate (1-E) (NT03-640,dmund Optics, Barrington, NJ) mounted horizontally for sup-ort. The inlet port of each of the two-way solenoid valves wasonnected to a 4 L solvent bottle (eight 4 L solvent bottles total)ia high purity Teflon PFA (perfluoro alkoxy alkane) 318 mm

D × 159 mm ID tubing (1-F) (MN: 1920, Upchurch Scientific,ak Harbor, WA). The Teflon PFA tubing was coupled to the 4 Lottles through a custom designed two-port Teflon bottle cap (1-) (Biafore Cad Concepts, Pipersville, PA) via a nut and ferrule

ig. 1. Snapshot of the automated mobile phase preparation workstation.A) Normally closed (NC) solenoid valves, (B) custom valve manifold, (C).6 m × 0.3 m base plate, (D) right angel brackets, (E) 0.6 m × 0.3 m base plate,F) Teflon PFA 0.318 cm OD × 0.159 cm ID tubing, (G) two-port Teflon bottleap, (H) gas regulator, (I) telescopic solvent delivery manifold, and (J) analyticalalance.

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r. A 1141 (2007) 182–190 183

P-308W & P-300X, Upchurch Scientific, Oak Harbor, WA).sing the second port of the bottle caps, the solvent reservoirsere connected to a 10-way gas manifold (not shown in Fig. 1)

5469K173, McMaster-Carr, New Brunswick, NJ) via TeflonFA tubing. A positive pressure was applied to the headspace ofach of the eight 4 L solvent bottles with nitrogen gas (or othernert gas) (≈96.5 kPa) from an in-house gas source. A gas regu-ator (1-H) (R352-02ASS, Watts Fluidic Inc., Kittery, ME) wassed to control the gas pressure. To prevent over pressurizationf the solvent bottles, one port of the 10-way gas manifold wastted with a pressure release valve set at 138 kPa (not shown inig. 1) (9889K204, McMaster-Carr, New Brunswick, NJ).

The outlet port of each of the solenoid valves was con-ected to a solvent delivery tube Teflon PFA tubing (158 mmD × 318 mm OD × 77.5 cm long) (1640, Upchurch Scientific,ak Harbor, WA) that was fed into a custom designed tele-

copic solvent delivery manifold (1-I) (Biafore CAD Concepts,ipersville, PA). This solvent delivery manifold was suspendedrom the custom valve manifold and centered above an analyticalalance (1-J) (E1F120, Ohaus, Pine Brook, NJ). The telescopicolvent delivery manifold was height adjustable allowing sol-ent collection vessels of various volumes and dimensions to besed. The analytical balance served as a real time data feedbackoop during solvent delivery for the computer control logic as alluantitative solvent delivery was mass based. Computer controlf the solvent delivery process was accomplished using RS-32 communication (FP-1000, National Instruments, Austin,X), a Labview-based control logic (Process Automation, Belleead, NJ), and a relay fieldpoint module (FP-RLY-420, National

nstruments, Austin, TX).

. Computer control logic and data base

The workstation’s control software (Process Automation,elle Mead, NJ) was written using Labview 7.0 (National Instru-ents, Austin, TX) and is accessible as a graphic interface. For

peration, the mobile phase is first defined using a series ofull down menus (Fig. 2) from the Components list. The sys-em can be configured with up to eight different solvents forse in the preparation of a single mobile phase. After selectinghe desired mobile phase constituents, the volume percentagef each component must be defined as well as the total volumef mobile phase to be prepared (range: 10 mL–4 L). In additiono the above stated information, a descriptive mixture name andhe name of the individual operating the workstation must bentered into the predefined fields of the control software beforehe automated preparation can begin.

Since all solvent dispensing is metered using an analyti-al balance, a database containing the densities of commonlysed mobile phase constituents was incorporated into the work-tation’s software. This database is user accessible allowingdditional stock solvents and their densities to be added aseeded. For compliance purposes, a second database (Stock

olvent Database) (Fig. 3) was incorporated into the soft-are to record and track information about each stock solvent.uch information recorded includes the solenoid valve used forelivery, solvent name, CAS number, manufacturer, lot num-

184 K. Swinney et al. / J. Chromatogr. A 1141 (2007) 182–190

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er, expiration date, date the stock solvent bottle was opened,nd safety warnings/hazards. This information is added to theatabase by the user and updated each time the stock solvent iseplenished/replaced, and also incorporated into the final reportetailing the specifics of each mobile phase preparation.

Once all of the above stated information is entered into theontrol software, the system is ready to begin the preparationf the requested mobile phase. Since the control software wasesigned to dispense only one mobile phase component at aime, a filling sequence is generated based upon the order the

obile phase ingredients appear in the components list (orderelectable by the user) (Fig. 2).

Each dispensing routine begins with the control software sig-aling the analytical balance to tare the collection vessel. Thenhe target dispense volume of component 1 is determined by mul-iplying the requested volume percentage by the total volume of

obile phase desired. The mass set point of component 1 ishen established by multiplying the calculated dispense volumey the solvent’s density.

After the set mass point for component 1 is attained within

1% error range, the final mass reading is recorded from the

nalytical balance in the mobile phase preparation report as thectual mass of component 1 delivered. Using component 1’sctual mass and density, the actual volume delivered is then cal-

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tion’s user interface and control logic software.

ulated and included in the preparation report along with the totalispense time for component 1. The complete process begin-ing with taring of the balance is then repeated for all otheromponents of the mobile phase. After the mobile phase is pre-ared, a preparation report (Fig. 4) is issued as a label for theobile phase vessel, a report for the notebook, and a data file

hat is stored as raw data. A typical 2 L mobile phase will require10 min to prepare.

. Chemicals and reagents

All chemicals and reagents used in this project were obtainedhrough Sigma–Aldrich Chemical Company (St. Louis, MO)nd were analytical grade or better; all of the organic solventsere obtained through VWR Scientific (West Chester, PA) andere HPLC grade. The deionized water was obtained in-house

rom a Millipore water filtration unit (Academic, Millipore, Bil-erica, MA).

. Solvent dispensing as a function of solvent delivery

ube diameter

Using deionized water, a positive pressure of 83 kPa appliedo the solvent bottles, and an Omega two-way normally closed

K. Swinney et al. / J. Chromatogr. A 1141 (2007) 182–190 185

Fig. 3. User interface for the stock solvent database.

Fig. 4. Mobile phase preparation report/label.

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olenoid valve (CV = 0.14) (SV-1211, Omega, Stamford, CT),he workstation was instructed to dispense 500 mL of water usingolvent delivery tubes of various inner diameters (0.050, 0.0762,.102, and 0.158 cm) and 77.5 cm in length. A total of three trialsere completed for each tube inner diameter. The time required

or the workstation to dispense the target volume as well as theccuracy and precision of solvent delivery was recorded by theorkstation’s control logic and database.

. Solvent dispensing as a function of applied pressure

Using deionized water and a solvent delivery tube 77.5 cmong with an inner diameter of 0.158 cm, the positive pressurepplied to the solvent bank was varied (83, 96, 110, and 124 kPa).he workstation was instructed to dispense 500 mL of watert each applied pressure and the time required for the systemo reach its target dispense volume was recorded along withhe accuracy and precision of the delivery by the workstation’somputer control logic and database. A total of three trials wereompleted at each pressure.

. Solvent dispensing as a function of valve CV

Two solenoid valves of different flow coefficients (CV)0.14 (SV-1211, Omega, Stamford, CT) and 0.047 (South Bendontrol Inc., South Bend, IN), respectively] were selected toetermine the affect of CV on dispensing accuracy and preci-ion while other variables were kept constant. CV is defineds the flow rate through an opened valve (gallons per minute,pm) divided by the square root of the pressure drop (psi)cross the valve [3]. An applied pressure of 96 kPa was selectedo pressurize the solvent bank and a 0.158 cm ID × 0.318 cmD × 77.5 cm long solvent dispensing tube was chosen. Theispensing accuracy and precision of the workstation wasecorded for each solenoid valve for a dispense volume of00 mL of water. A total of five trials were completed for eachalve.

. Solvent dispensing accuracy and precision for a0 mL dispense

Using deionized water, a solvent delivery tube 77.5 cm longith an inner diameter of 0.158 cm, and a 96 kPa positive pres-

ure applied to the solvent bank, the ability of the workstation toispense 10 mL was determined. The workstation was instructedo dispense 10 mL of water for a total of 24 trials. The accuracynd precision of the delivery was recorded by the workstation’somputer control logic and database.

. Solvent dispensing as related to solvent viscosity andurface tension

Using a solvent delivery tube 77.5 cm long with an inner

iameter of 0.158 cm and a 96 kPa positive pressure applied tohe solvent bank, the workstation was instructed to dispenseL of acetonitrile. The time required for system to reach its

arget dispense volume was recorded along with the accuracy

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nd precision of the dispense by the workstation’s computerontrol logic and database. A total of five trials were completed.he same analysis was repeated using methanol and the results

rom the acetonitrile and methanol dispenses were compared tohe workstation’s dispensing statistics for 1 L of water.

0. Dispensing accuracy and precision of theorkstation as compared to manual preparation

Using a 1 L graduated cylinder, 4-laboratory scientists weresked to measure 1 L of water using a tared graduated cylinderitting on an analytical balance. The mass of water dispensed intohe graduated cylinder was recorded. Each scientist completedtotal of five trials. The accuracy and precision of the four trialsere compared to the results produced by the workstation forispensing five 1 L volumes of water.

1. Generation of a HPLC mobile phase

The automated mobile phase preparation workstation wassed to prepare five 1 L mobile phases consisting of 60:40 ace-onitrile:water. The mass of acetonitrile and water dispensedy the workstation was recorded for each preparation. Simul-aneously, five 1 L mobile phases (60:40 acetonitrile:water)ere manually prepared. Each mobile phase was then testedy HPLC (Agilent 1100, Agilent Technologies, Palo Alto,A) using a column test mixture (methyl, propyl, and butyl-araben in 60:40 acetonitrile:water), a 150 mm × 4.6 mm, 5-�m,henomenex Luna C18 column (00F-4041-E0, Phenomenex,orrance, CA), and absorbance detection at 254 nm. The reten-

ion times of each component of the test mix was recorded andhe results from all analyses (five manually prepared mobilehases plus the five workstation prepared mobile phases) wereompared.

2. Results and discussion

An automated mobile phase preparation workstation capablef preparing 50 L of mobile phase during a standard businessay (8 h in a day leads to 6.25 L/h) while meeting the necessaryequirements of safety, accuracy, precision and documentationas designed, tested, and implemented.The performance (precision, accuracy, and time for solvent

elivery) of the automated mobile phase preparation worksta-ion was optimized with respect to the dispensing tube inneriameter, applied pressure and valve CV. To study the effect ofhe dispensing tube’s inner diameter on the workstation’s dis-ensing accuracy and precision, the workstation was requestedo aliquot 500 mL of water using an Omega two-way normallylosed solenoid valve while the inner diameter of the dispensingube (0.0508, 0.0762, 0.1016, and 0.158 cm) was varied. Theength of each dispensing tube was set to 77.5 cm and a positiveressure of 83 kPa was applied to the solvent bank. By setting

he applied pressure and length of the dispensing tube constant,he resultant flow rate produced in each tube should be directlyelated to the dispensing tube’s inner diameter as defined by theagen–Poiseuille equation [4]. For fluid flow in an open tube,

K. Swinney et al. / J. Chromatogr. A 1141 (2007) 182–190 187

Table 1Dispensing accuracy and precision as a function of dispensing tube size

Tube diameter (cm) Dispensed volume (mL) Accuracy (% error) Precision (% RSD) Dispense time (min)

0.050 500.19 0.04 0.02 24.29 ± 0.140.076 500.10 0.02 0.08 9.74 ± 0.210.102 500.50 0.10 0.06 4.73 ± 0.130.158 500.50 0.10 0.09 3.02 ± 0.16

Table 2Dispensing accuracy and precision as a function of applied pressure

Applied pressure (kPa) Flow rate (mL/s) Dispensed volume (mL) Accuracy (% error) Precision (% RSD) Dispense time (min)

83 3.61 ± 0.05 500.50 0.10 0.06 4.73 ± 0.1396 4.13 ± 0.05 500.73 0.15 0.06 2.76 ± 0.18

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ncreasing the inner diameter of the dispensing tube should resultn a higher flow rate or solvent dispensing rate if all other vari-bles are held constant. Realizing that the increased dispenseate may affect the accuracy and precision of solvent delivery,hree 500 mL dispensing routines were completed for each tubenner diameter. Reported in Table 1 are the results from this dis-ensing experiment. Increasing the tubing diameter from 0.0508o 0.158 cm had a sizeable effect on the accuracy and the pre-ision of the workstation’s solvent delivery. However, the lossn accuracy/precision associated with using the larger diameterispensing tube was negligible when the dispensing time associ-ted with each tube inner diameter was considered. Therefore, inhe interest of minimizing the time required for solvent delivery,0.158 cm ID solvent dispense tube was chosen for all future

xperiments.Next using the 0.158 cm inner diameter solvent dispense tube

nd the Omega two-way normally closed solenoid valve, theolvent bank pressure was varied and its effect on flow ratend dispensing accuracy and precision was investigated. Theositive pressure applied to the solvent bank was varied from3 to 124 kPa in 13 kPa increments while the workstation wasnstructed to dispense 500 mL of water 3 times for each appliedressure. As shown in Table 2, the flow rate was shown toncrease nominally (see Table 2) with increased pressure onhe solvent bank, as expected from the Hagen–Poiseuille equa-ion [4], and resulted in an overall nominal decrease in dispenseime. The flow rate does not increase to the level suggested byhe equation due to other factors such as flow restriction fromhe valves and other hardware. It should be noted that the dis-

ense time is not only a function of flow rate but is also affectedy the number of times the computer control logic must acti-ate/deactivate the solenoid valve in order to reach the targetispense volume.

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alve CV (gpm/psi) Dispensed volume (mL) Ac

mega 0.14 500.73 0.1outh Bend 0.047 500.99 0.2

0.24 0.08 2.72 ± 0.370.14 0.08 2.64 ± 0.09

The workstation’s ability to accurately and precisely dis-ense a specified volume of solvent was also determined atach tested pressure. The workstation’s dispensing accuracy wasound to slightly decrease with increasing pressure (see Table 2).lthough the decrease in dispensing accuracy associated with

he 124 kPa solvent bank pressure was minimal, the reduction inispense time was not large enough to justify pressurizing theolvent bank at the higher pressure. In the interest of safety, itas decided that the solvent bank should be pressurized at the

owest possible pressure without altering the performance of theorkstation. Therefore, an applied pressure of 96 kPa was cho-

en as the optimal solvent bank pressure and used for all furtherxperiments. To further ensure safe operation of the workstationnd prevent over pressurization of the solvent bank, a pressureelief valve set at 138 kPa was incorporated into the gas manifoldnd the entire workstation was housed in a standard explosionroof laboratory ventilation hood.

Upon optimization of the dispensing tube inner diameter andhe positive pressure applied to the solvent bank, the effect ofhe solenoid’s valve flow coefficient (CV) on the workstation’sispensing performance was studied. To determine the effect CVas on solvent dispensing accuracy and precision, 2 two-wayormally closed solenoid valves were chosen. The first valvehosen was the Omega two-way normally closed solenoid valveith a CV value of 0.14. The second valve chosen was the Southend two-way normally closed solenoid with a CV value of.047. Here, 5–500 mL volumes of water were dispensed by theorkstation for each valve. Using the data capture feature of

he computer control logic, the accuracy and precision of the

ispensing routine for each valve was determined. As shown inable 3, the CV of the valve (at least in the range studied) doesot appear to have a great effect on the workstation’s dispensinguality. Therefore, due to better chemical compatibility of the

curacy (% error) Precision (% RSD) Dispense time (min)

5 0.06 2.76 ± 0.180 0.06 2.78 ± 0.08

188 K. Swinney et al. / J. Chromatogr. A 1141 (2007) 182–190

Table 4Dispensing accuracy and precision as a function of viscosity and surface tension

Solvent Viscosity (cP) Surface tension (dyne/cm) Dispensed volume (mL) Accuracy (% error) Precision (% RSD) Dispense time (min)

Water 1.00a 72.8a 1000.16 0.02 0.02 7.56 ± 0.15Acetonitrile8 0.38b 19.10a 1000.15 0.02 0.01 5.64 ± 0.26M

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ethanol8 0.55a 22.5a 1000.29

a Value measured at 20 ◦C.b Value measured at 15 ◦C.

thylene Propylene Diene Monomer (EPDM) seal in the Southend solenoid valve with organic solvents commonly used inPLC, the South Bend solenoid valve (valve with the smallerV) was chosen for future experiments.

With the workstation designed for dispensing volumes upo 4.5 L for the preparation mobile phases, it was necessary toefine the minimum volume the system would be allowed to dis-ense for a single component of a mobile phase and determinehe dispensing accuracy and precision. From experience gainedith the system and to ensure compliance with the standard spec-

fication set by ASTM International for Class A glass graduatedylinders, a minimum dispense volume of 10 mL was selected.sing the optimized instrumental configuration (i.e. 0.158 cm

D diameter dispensing tube, positive solvent bank pressure of6 kPa, and the South Bend two-way normally closed solenoidalve), the ability of the workstation to dispense 10 mL of wateras assessed by instructing the workstation to dispense 10 mL

or a total of 24 trials. The computer control software recordedhe dispensed solvent mass, and the accuracy and precision ofhe 24 dispenses was determined. For the 24 dispenses, the aver-ge volume delivered was 9.97 ± 0.06 mL, which correspondso a dispensing accuracy of 0.35% and a dispensing precision of.63%, which exceeds the specifications (tolerance = ±0.1 mL)et by the ASTM for a 10 mL Class A graduated cylinder [5]. Ithould be noted that the label/preparation report always reportshe true concentration of each component of the mobile phasend that the end user makes the final decision whether to acceptr reject a mobile phase.

Next, the workstation’s dispensing accuracy and precisionas determined for solvents with lower viscosities and surface

ensions than that of water (viscosity = 1.00 cP at 20 ◦C; surfaceension = 72.8 dyne/cm at 20 ◦C) [6]. Acetonitrile and methanolere chosen as the organic solvents for this study since they

re the most commonly chosen solvents for the preparation ofPLC mobile phases in our labs. To determine the workstation’sispensing accuracy and precision for methanol and acetonitrile,he system was instructed to dispense 1 L of the solvent 5 timesor each solvent. The flow rate, dispense time, as well as, the dis-ensed mass was electronically captured by the computer contrologic and used to calculate the system’s dispensing performanceTable 4). For acetonitrile, the 5 trial dispensed mass averageor a 1 L dispense routine for acetonitrile (d = 0.7899 g/mL)esulted in a dispense error of only 0.02%. While for methanold = 0.7866 g/mL), the 5 trial dispense routine resulted in a dis-

ense error of 0.03%. Comparison of the workstation’s ability toispense water versus solvents of lower surface tensions and vis-osities, such as acetonitrile or methanol, shows no significantoss in dispensing performance of the workstation was observed.

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With the automated mobile phase preparation workstation’sonfiguration and performance optimized, the ability of the sys-em to accurately dispense a target volume was compared to

anual methods using a graduated cylinder. Here four labora-ory scientists were asked first to dispense 1 L of water using a000 mL graduated cylinder. Each scientist repeated the proce-ure five times and the results are shown in Fig. 5 along withcomparison to the workstation’s performance. For illustrationurposes, all recorded dispensed masses were converted to vol-mes using the density of water (0.9971 g/mL at 25 ◦C). Ashown in the graph, the workstation was not only more accu-ate in its delivery of the target volume of 1 L of water but alsoore precise. For example, the 5 trial average for the work-

tation’s delivery was 1000.65 ± 0.47 mL (% error = 0.07%),hile the most accurate human trial had a 5 trial average of99.33 ± 2.28 mL (% error = 0.23%). As shown in Fig. 5, theccuracy and precision of the 4 laboratory scientists to measureL of water using a 1000 mL, Class A, graduated cylinder varied

ignificantly.As a final test of the automated mobile phase preparation

orkstation, the system was instructed to prepare five 1 L quan-ities of a 60:40 acetonitrile:water mobile phase. Each mobilehase was then used to perform an isocratic HPLC separationf a column test mixture (methyl, propyl, and butylparabenach present at a concentration of 10 mg/mL in 60:40 acentoni-

ig. 5. Comparison of the dispensing accuracy and precision of the automatedobile phase preparation workstation with that obtained manually by five sci-

ntists using a 1 L graduated cylinder for the dispensing of 1 L of water.

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hase 30 min before the analysis, respectively. Using a samplenjection of 1 �L, the test mix was analyzed in triplicate.

In order to compare the chromatographic results obtainedith the mobile phase prepared by the workstation to thosebtained with a mobile phase prepared manually, five 1 L vol-mes of 60:40 acetonitrile:water mobile phase were preparedy a laboratory scientist using a graduated cylinder. The col-mn test mix was then analyzed in triplicate using each of theve manually prepared mobile phases. Shown in Fig. 6 are twohromatograms. One is a representative chromatogram of theest mix analysis using the mobile phase prepared by the work-tation. The second is a sample chromatogram obtained for theolumn test mix using one of the manually prepared mobilehases. As shown by the retention times reported on the chro-atographs, there is no significant difference between the two

hromatograms suggesting that the workstation is capable ofreparing mobile phases for HPLC that are as robust as thoserepared manually. In fact, for the 50 chromatograms obtainedsing the manually prepared mobile phases, the retention

ig. 6. Comparison of the chromatographic results obtained using a manu-lly prepared mobile phase (A) vs. a mobile phase prepared by the automatedobile phase preparation workstation (B) for the separation of a paraben col-

mn test mixture. Sample: methyl, propyl, and butylparaben test mix in 60:40cetonitrile:water each at a concentration of 1 mg/mL. Mobile phase: 60:40 ace-onitrile:water. Detection: UV absorbance at 254 nm. Injection volume: 1 �L.low rate: 1 mL/min. Column: Phenomenex Luna 150 mm × 4.6 mm, C18,�m. Peak 1: methylparaben, Peak 2: propylparaben, Peak 3: butylparaben.

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r. A 1141 (2007) 182–190 189

imes were reported to be 2.23 ± 0.01 min for methylparaben,.32 ± 0.01 min for propylparaben, and 4.36 ± 0.03 min forutylparaben. These retention times are almost identical to theetention times obtained for the methyl, propyl, and butyl-araben test mix analyzed with the workstation prepared mobilehases (2.22 ± 0.00 min for methylparaben, 3.29 ± 0.00 min forropylparaben, and 4.32 ± 0.00 min for butylparaben). The sim-larity between retention times indicates that the automated

obile phase preparation workstation is capable of preparingobile phases that can be used in HPLC analyses without

acrificing or altering the performance of the separation as com-ared to the same analysis utilizing manually prepared mobilehases.

3. Conclusion

An automated mobile phase preparation workstation capablef dispensing volumes ranging from 10 mL to 4.5 L of mobilehase was designed and tested. The system’s performance wasptimized with respect to liquid transfer tubing inner diame-er, pumping pressure, valve CV, and computer control logic.

ith just under a 6-min preparation time for a 1 L mobile phase,he business need of preparing 50 L of mobile phase in oneormal day of operation was not only met but also exceededcapable of preparing 80 L of mobile phase in an 8 h period).he workstation was shown to possess excellent solvent deliv-ry accuracy (% error ≤ 0.35% for a target solution volumef 10 mL (tolerance = ±0.06 mL) and precision (RSD ≤ 0.63%or a target solution volume of 10 mL) while improving uponhe capability of manual preparation using a graduated cylin-er (tolerance = ±0.1 mL [5]; % error = 1.0%). Furthermore, theispensing precision and accuracy of the system was found toe unaffected by the various viscosities and surface tensions ofommonly used HPLC solvents. Also with the electronic dataapture capability of the computer control logic and database,afety and regulatory compliance was ensured through detailedocumentation of the mobile phase preparation and mobilehase label generation. While the system cannot be used to pro-uce buffered mobile phases, it may be possible to modify theystem to add this capability in the future.

cknowledgements

The authors would like to acknowledge the following peo-le for their support and contribution to the project: Cynthiaaryanoff, James V. Weber, Alan Oyler, Greg Worsila, Hen-

ik Rasmussen, Bruce Weber, Ron Muenz, Ronnie McDowell,avid Mitchell and Justin Kraus. The authors would also like to

hank Michael Biafore of Biafore CAD Concepts, Lew Drakef Process Automation and the Penn State University’s Co-Opffice.

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