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UV Visible Spectroscopy for Quantification of Drop-on-Demand Inkjet Performance Amin Famili, William J. Baldy, Jr., and Saurabh A. Palkar* Cordis Corporation, a Johnson & Johnson Company, Welsh and McKean Roads, Spring House, Pennsylvania 19477, United States ABSTRACT: Drop-on-demand inkjet technology offers a method through which nano- to microgram quantities of a substance can be delivered to a precise target location. However, few measurement techniques exist to quantify the material dispensed in quantities representative of those deposited on a typical target. While many methods exist that are suited to quantitate the cumulative material dispensed by large numbers of drops, few are capable of adequately quantifying subthousand drop counts. This gap in the quantitation methodology is significant since typically the first few drops in a burst have different mass from that of the subsequent drops. A UV visible spectroscopic method is presented here as a means to quantify the dispensed material associated with drops numbering in the few tens to hundreds, with this method exhibiting excellent repeatability and reproducibility without the need for lengthy calibrations or sample preparation times. The resolution and precision of this method make it an attractive choice for inprocess testing, in which both accuracy and throughput are of high importance.
’ INTRODUCTION In an increasingly large number of industries, the ability to accurately and repeatedly deposit nanogram quantities of a given substance is critical to the development of new technologies.1 4 This is largely driven by a move toward micro- and nanoscale products that require extremely accurate processing steps. Many applications require repeatable deposition of nano- or picoliter quantities of solutions to precise locations on a target. This is particularly true in the manufacturing of many medical devices5 where the amount and location of drug loading must be controlled to very precise specifications. In such cases, dropon-demand inkjet technology is an attractive choice as it addresses the needs for both accurate targeting and repeatable droplet ejection. Particularly for these kinds of highly controlled applications, the quantity of substance being ejected from the inkjet devices must be known to extreme accuracy. Various methods have been described to this end in previous publications including the use of atomic force microscopy cantilevers,6 quartz crystal microbalances,7,8 nanomechanical resonators,9 and gravimetry.10 However, all of these methods require either highly sophisticated instrumentation, time-consuming calibration processes that are impractical for a manufacturing process application, or a large number of drops11 to ensure an accurate measurement. A system that can quantify the material dispensed in small drop numbers, requires little calibration, and can be easily integrated into an existing process is of general interest. UV visible spectroscopy meets these criteria due to its sensitive detection limits, relatively simple calibration, and short sampling time. Despite UV visible spectroscopy’s relative weakness in identifying unknown compounds, its ability to quantify known substances in solution is quite robust.12 As such, it is a common choice for applications in which a known substance is dissolved in a known solvent and only determination of the concentration is desired. Further, in mixed solutions with more than one component, absorbance values measured at multiple wavelengths can be compared to determine relative concentrations of individual components, which is useful r 2011 American Chemical Society
for assessments of solution mixing and degradation of individual components. UV spectroscopy has been used in the past11 to quantitate amounts of drugs dispensed by inkjet devices. The novelty of our work described here is that we use a different solvent (from the one used to make inkjet solution) to collect drops ejected by the inkjet, and then monitor the absorbance related to the solvent used to make inkjet solution. Since the solvent makes up more than 80% of the weight of each drop of solution dispensed by the jet, only a few drops are needed to get a good signal-to-noise ratio. Thus average drop mass can be determined by using only a few tens to hundreds of drops (this is what is typically delivered to targets of interest in many applications) instead of thousands of drops needed in the previously published work. It is extremely important to have a method that allows quantitation of the same number of drops that are delivered to the target during the application of interest. The reason for this requirement is that typically the first few drops in a burst have different mass from that of the subsequent drops.10,13 For example, if only 20 drops are deposited at the target but 10 000 drops are used to calculate the average drop mass during calibration, then there will be an offset between calibration and actual application. Development of a UV visible spectrometry method suitable for quantitation of droplets (few tens to hundreds) ejected from an inkjet device is described in the following sections. Such a method is not only beneficial for research and development purposes but is also suitable for manufacturing environments, allowing for immediate, accurate measurements of inkjet behavior. Further, this method’s use of nonvolatile solvents for sample collection ameliorates problems associated with measurement of volatile solutions, as are often used in inkjet applications. Received: November 23, 2010 Accepted: July 19, 2011 Revised: July 15, 2011 Published: July 19, 2011 9829
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’ MATERIALS AND METHODS The general principle behind this method involves dispensing a known number of droplets of a solution of interest from the inkjet dispenser (herein referred to as the inkjet solution) into a cuvette loaded with a precise amount of a collection solvent (which is not used in the inkjet solution). An absorbance spectrum of the sample in the cuvette (herein referred to as the spectroscopic solution) is then measured, and a previously determined standard calibration curve is used to calculate the concentrations of the various components in the cuvette. The resultant concentrations can then be used to calculate the mass of the droplets dispensed from the inkjet since the original volume of the collection solvent in the cuvette is known. The drop-on-demand apparatus used in this experiment was a low temperature biologics inkjet (MJ-AB-63-40, MicroFab Technologies, Plano, TX, USA) with a 40 μm orifice diameter. This device was controlled via a MicroFab JetDrive III electronics control unit connected to a standard computer, through which electrical parameters for the driving waveform were configured. The inkjet solution used in these studies to create droplets was composed of poly(lactic-co-glycolic acid) (PLGA) and sirolimus (rapamycin) dissolved in dimethyl sulfoxide (DMSO). An Agilent 8453 UV visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) was used to measure absorbance data which were subsequently analyzed using the Agilent ChemStation software suite. Deionized water (Milli-Q or equivalent) was used as the collection solvent loaded into the cuvettes for all samples in this study due to its low cutoff wavelength and its ease of transport and disposal in a manufacturing environment. Samples were prepared and collected in glass scintillation vials and then transferred using a pipettor to an Agilent quartz cuvette (10 mm path length). This cuvette required a sample volume of 1 mL, so all samples described herein were dissolved in this volume of water. Absorbance values were collected between 190 and 1100 at 1 nm increments with a 1 s integration time. Preliminary screening experiments were conducted to determine the feasibility of measuring the components in solution at various concentrations, so absorption was measured over a wide range of wavelengths. Later experiments used absorbance values only at a wavelength of 208 nm, corresponding to the peak absorbance for DMSO. When more precise absorbance determinations were required, a cuvette was first tared on an analytical balance (Mettler Toledo XP205 DeltaRange) and then loaded with 1 mL of deionized water and weighed on the same balance. This allowed for more accurate determination of concentration since the solvent volume was determined both volumetrically and gravimetrically. The water used for these experiments was also sonicated for 1 min to remove any dissolved gases just prior to UV measurements. This method is not restricted to drugs/materials soluble in DMSO. The only requirement is that the solvent used to make the inkjet solution (in our case DMSO) should be miscible with the collection solvent (in our case water) and have a UV absorption peak. ’ RESULTS AND DISCUSSION This study was designed for the development of a UV visible spectroscopic method for characterization of inkjet microdispensers. Of interest were two primary goals: to improve upon the detection limits of current methods by being able to quantify
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Figure 1. UV visible spectra of various concentrations of DMSO dissolved in deionized water. Spectra shown after subtracting water blank.
inkjet output in the tens to hundreds of drops instead of thousands and to aid in the assessment of inkjet solution mixing properties after ejection. The first goal required the tracking of a single species in the spectroscopic solution and measuring absorbance while carefully controlling for other factors. The second goal was an extension of the first and sought to track multiple species in the spectroscopic solution to ensure homogeneity and gain insight into possible inkjet-induced nonuniformities such as demixing and precipitation. Screening Studies. Initial feasibility experiments sought to determine whether the substances in the inkjet solution were detectable and whether the associated concentrations would yield absorbance values adequate to quantify the species in question without detector saturation. To accomplish this, a wide range of spectroscopic solution concentrations were measured in the wavelength range of 190 1100 nm using solutions of only DMSO in water. As DMSO constituted 86% of the inkjet solution by volume, it was postulated that this would exhibit the strongest absorbance and would constitute the species of greatest impact in determining jet output. Figure 1 shows the first of such experiments, in which various concentrations of DMSO were dissolved in deionized water (0.1 μg/mL 100 μg of DMSO/mL of water). This concentration range corresponds to about 1 to 1000 drops of DMSO/mL of water if the inkjet drop size was around 100 ng or to about 10 to 10000 drops of DMSO/mL of water if the inkjet drop size was around 10 ng. (Please note that the inkjet was not used to create Figure 1.) Concentrations between these values produced an absorbance peak at 208 nm, within the desired absorbance range for the spectrometer, with this wavelength coinciding with the theoretical absorbance of DMSO.14 Despite the proximity of the absorbance peak associated with DMSO to the cutoff wavelength of water, the peak was distinct enough to justify further investigation. The use of a solvent (used to make the inkjet solution) as the species of interest for quantitation introduces problems associated with evaporation (and/or moisture absorption for hygroscopic solvents like DMSO) as this will hinder this method’s ability to accurately assess the actual amount of substance being ejected from the inkjet. While many applications would need to take this into account, this is of little concern for the method being developed here for the following two reasons. First, after ejection from the jet the drop spends very little time (10 ms or less) in the air before reaching the collection solvent in the cuvette. Second, once the drop reaches the collection solvent, 9830
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Figure 2. UV visible absorbance spectrum of a 10 μg/mL solution of inkjet solution (DMSO, sirolimus, and PLGA) in deionized water. Absorbance peaks were found to occur at 208 nm, corresponding to DMSO and PLGA, and a triplet centered at 280 nm, corresponding to sirolimus. Both DMSO and sirolimus peaks exhibit absorbance in the desired range for accurate quantitation, while the concentration of PLGA was below the quantitation limit and thus did not contribute to the peak at 208 nm.
what matters is the evaporation of the solution in the cuvette, which is minimal since water is used here as the collection solvent in the cuvette. Having determined the ideal concentration range for the primary component of the inkjet solution, drug and polymer were added to this solution and a similar screening study was conducted to determine absorbance peaks associated with these other species. A typical spectrum for the full solution is shown in Figure 2. In addition to the absorbance peak at 208 nm associated with DMSO and PLGA, a triplet peak also exists centered at 281 nm with shoulder peaks at 271 and 293 nm. This triplet matches the theoretical absorbance wavelength and behavior of sirolimus15 but exhibits an absorption maximum approximately one-third of the absorbance of DMSO. However, since the absorbance peaks of both species can be captured at the same spectroscopic sample concentration, simultaneous assessments of concentrations for these two species are feasible. While both DMSO and PLGA absorb at 208 nm, the concentration of PLGA in the spectroscopic solution was below the detection limit and as such did not contribute to the absorbance at this wavelength. Evaluation of Operating Region. In order to refine this method for maximum sensitivity and repeatability, spectra were analyzed further for statistical robustness. Of concern for a suitable UV visible spectroscopic method are both linearity of concentration responses and repeatability of sample readings at a given wavelength.16 These were assessed by determining (a) the linearity correlation coefficient for absorbance versus concentration at a range of concentrations bracketing the desired drop numbers and (b) the standard deviation of absorbance readings taken of the same sample across all available wavelengths. A properly designed spectroscopic method will be optimized at a wavelength at which linearity is maximized and sampling variability is minimized. The results of these analyses are shown in Figures 3 and 4. The analysis for linearity yielded a broad range of wavelengths between 195 and 295 nm that exhibit near-perfect correlation between absorbance and concentration. The absorbance peak at 208 nm associated with DMSO falls in the middle of this region, indicating a robust operating space surrounding this peak. The analysis of repeatability yielded an absolute minimum in variability
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Figure 3. Correlation coefficient versus wavelength for a standard curve with DMSO concentrations ranging from 0.1 to 20 μg/mL. Coefficients near 1.0 are exhibited between wavelengths of 195 and 295 nm.
Figure 4. Repeatability, expressed as % RSD, versus wavelength for 10 absorbance readings of the same sample at a nominal concentration of 10 μg/mL.
at 208 nm, further confirming that this wavelength is best suited for quantitation of droplet size. Calibration Curve, Accuracy, and Precision. Figure 5 shows a calibration curve constructed at the wavelength of 208 nm. Three samples were tested at each concentration to generate this plot. Linearity was found to be excellent (R2 of 0.997) up to 10 μg/mL concentration. The accuracy was studied using known amounts of DMSO up to 10 μg/mL, finding their absorbance, using the calibration curve to calculate DMSO concentration in the cuvette, and then comparing known versus calculated concentration. For DMSO concentrations from 0.1 to 10 μg/mL (i.e., from 100 ng to 10 μg of DMSO in the cuvette, which corresponds to 1 100 drops if the average drop mass is around 100 ng or 10 1000 drops if the average drop mass is 10 ng, etc.), the difference between the known and the calculated concentrations was 1% or less. Below 0.1 μg/mL the differences between the two were found to be larger than 1% and we are trying to further refine the method for these really low concentrations. 9831
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Figure 5. Standard calibration curve at 208 nm generated by dissolving various concentrations of DMSO in water.
Having established an appropriate concentration and wavelength at which to quantify the components of the inkjet solution, an ANOVA Gage Repeatability and Reproducibility study17 was designed to assess the relative impacts of the measurement technique and sample-to-sample differences. The results of this study indicated that the greatest contribution to differences in absorbance was driven by actual sample-to-sample differences with total variation (reproducibility plus repeatability) for a given sample being less than 1%. The calibration curve in Figure 5 was used to determine the mass of drops ejected by inkjet as follows. One milliliter of sonicated water was placed in a cuvette, then the desired number of drops was placed in this cuvette using the inkjet, and the absorption at 208 nm was determined; the calibration curve in Figure 5 was then used to back calculate the concentration in the cuvette, hence the drop mass. Sample Stability. Spectroscopic methods can be especially susceptible to sample degradation, resulting in heightened sensitivity to the age of the sample. To quantify this effect for the current method, a number of samples at various concentrations were measured over a period of 5 h in order to track their degradation. Results indicated that sample degradation was concentration dependent with absorbance values decreasing at a rate of approximately 2.0% per hour at a nominal spectroscopic sample concentration of 8 μg/mL. This indicates that samples quantified using this method degrade over time, but this degradation is not significant if they are analyzed within 1 h after sample collection. Comparison with Other Methods. As mentioned in the Introduction, the purpose of this work was to come up with a simple technique for measuring the mass of drops ejected by inkjets with a focus on (1) simplicity, so the method can be used as an in-process test method on a manufacturing line, and (2) quantitating the number of drops relevant to the application (i.e., if 10 drops are being deposited at every target on a product, then the method should be able to collect 10 drops and quantitate their mass. Previous use of UV spectroscopy11 needed thousands of drops to determine average mass since the absorbance spectra of the dissolved drug was used for quantitation. The novelty of our method is use of the absorption spectra of the solvent used to make inkjet solvent; this allows quantitation in the range of 100 ng 10 μg of inkjet solution deposited in the cuvette
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(so 1 100 drops if average drop mass is around 100 ng, or 10 1000 drops if the average drop mass is 10 ng, etc.). The method exhibits really good linearity and precision in this mass range, allowing detection of drop mass with an uncertainty of around 1%. As stated in the Introduction, other methods (quartz crystal microbalance,6 atomic force microscopy,6 gravimetry,10 etc.) have been developed to quantitate drop mass ejected by inkjets. The gravimetry10 method developed by Verkouteren et al. offers a method superior to optical methods with total relative uncertainties in average droplet mass of about 1% when there is a continuous ejection rate of at least 14 ng/s or a burst aliquot of at least 72 μg. The UV method described here can work in lower burst aliquots (100 ng 10 μg) with similar accuracy and precision. The gravimetric method offers way of quantitation in the picogram range (when continuous ejection is used), similar to the quartz crystal microbalance. The latter requires application of specific careful calibration; otherwise the accuracy is not good. The main advantage of the method described here is its simplicity and robustness. UV spectroscopy is a routine technique in analytical laboratories, and it does not need fancy equipment, software, or a controlled environment. Hence it is possible to use this technique as an in-process method at the manufacturing setting to quickly determine the average drop mass for bursts in the 100 ng 10 μg range and then select the appropriate number of drops to reach the mass target required for the application of interest.
’ CONCLUSIONS This paper has described the development of a UV visible spectroscopic method for quantitation of small samples of drops ejected from an inkjet microdispenser. Given the results detailed above, this method provides an excellent tool for assessing inkjet performance at bursts from 100 ng to 10 μg with total uncertainty around 1%. Further development of this method is ongoing with promising initial findings for quantifying jet performance below 100 ng using a smaller volume cuvette. This method also demonstrates the ability for quantitation of multiple species in solution simultaneously, allowing for assessments of solution uniformity after ejection. This method’s ease of setup, sample collection, and calibration makes it an attractive choice for in-process and research scenarios in which throughput is as important as accuracy. ’ AUTHOR INFORMATION Corresponding Author
*Tel.: (215) 628-5669. Fax (215) 540-4925. E-mail spalkar@ its.jnj.com.
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