Size-Exclusion Chromatography with Electrospray Mass Spectrometric

4 Size-Exclusion Chromatography with Electrospray Mass Spectrometric Detection William J. Simonsick, Jr. and Laszlo Prokai

Downloaded by SUFFOLK UNIV on January 22, 2018 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/ba-1995-0247.ch004

1

1

2

Marshall R & D Laboratory, DuPont, Philadelphia, PA 19146

Center for Drug Discovery, College of Pharmacy, University of Florida, Gainesville, FL 32610

2

We interfaced a size-exclusion chromatograph (SEC) to a mass spectrometer operating in the electrospray mode of ionization. Stable electrospray conditions were obtained using a tetrahydrofuran mobile phase containing ~10 M dissolved sodium salt, which affords pseudomolecular ions through cationization. Using the SEC with electrospray detection, we calibrated the SEC for an ethylene-oxide-based nonionic surfactant. The calibration standards were the surfactant oligomers themselves. The chemical composition distribution of acrylic macromonomers was profiled across the molecular weight distribution. A nonuniform chemical composition distribution was observed. Cross-linkers, additives and stabilizers, and coalescing solvents contained in a complex waterborne coating formulation were analyzed in a single experiment. -5

SIZE-EXCLUSION C H R O M A T O G R A P H Y (SEC) is a p o p u l a r technique used to obtain molecular weight distributions ( M W D ) on polymers and oligomers (I, 2). T r a d i t i o n a l l y , detection is accomplished b y a differential refractive index (DRI) detector. U n f o r t u n a t e l y , D R I provides little i n formation about the p o l y m e r c h e m i c a l composition. T h e use of mass spectrometry for detailed p o l y m e r analysis is b e c o m i n g more established due to the p r o l i f e r a t i o n of soft i o n i z a t i o n techniques that afford intact oligomer or p o l y m e r ions w i t h a m i n i m a l n u m b e r o f fragment ions ( 3 11). I n addition to the M W D information, the specific chemical structure, i n c l u d i n g e n d groups and the distribution of m o n o m e r units i n a cop o l y m e r , is obtained. F u r t h e r m o r e , the data furnished b y soft i o n i z a t i o n 0065-2393/95/0247-0041$12.00/0 © 1995 American Chemical Society

Provder et al.; Chromatographic Characterization of Polymers Advances in Chemistry; American Chemical Society: Washington, DC, 1995.


42

CHROMATOGRAPHIC CHARACTERIZATION OF POLYMERS

techniques are predictable. If a researcher hypothesizes about a given structure, the postulated structure has a molecular formula that i n addition to the nominal mass yields an isotope envelope that can be c o m pared w i t h the theoretical isotope envelope based on the molecular formula. Because soft ionization affords molecular or pseudomolecular ions, such hypothesis can be evaluated. Electrospray ionization mass spectrometry (ESIMS) is a soft ionization technique that has been w i d e l y applied i n the biological arena. O w i n g to its extremely low detection limits and its ultrasoft ionization process, E S I M S has been the most successful method of coupling a condensed phase separation technique to a mass spectrometer. U n d e r E S I conditions the sample l i q u i d is introduced into a chamber w i t h a h y podermic needle. A n electrical potential difference (usually 2 - 4 kV) between the needle inlet and the cylindrical surrounding walls promotes ionization of the emerging l i q u i d and disperses it into charged droplets. Solvent evaporation upon heat transfer from the ambient gas leads to the shrinking of the droplets and to the accumulation of excess surface charge. A t some point the electric field becomes high enough (up to 1 0 V/cm) to desorb analyte ions. This widely accepted desorption model (12) relies on the existence of preformed ions i n solution. In other words, the ions observed i n the mass spectra were originally present as i o n i z e d molecules i n solution. 9

Proteins and biopolymers are typically i o n i z e d through acid-base equilibria. L a r g e r biopolymers acquire more than a single charge. A charge envelope results from the analysis of a single species. Because mass spectrometers separate ions based on the mass-to-charge ratio, increasing the number of charges can be used to extend the operable mass range. In fact, F e n n and N o h m i (13) have observed polyethylene glycols w i t h molecular weights up to five million on a quadrupole mass analyzer w i t h upper mass limits of 1500 D a . Unfortunately, E S I M S has had limited applications on synthetic polymers (13, 14). U n l i k e biopolymers, many synthetic polymers have no acid or basic functional groups that can be used for i o n formation. M o r e o v e r , each species of a unique molecular formula can give rise to a charge distribution envelope, thus further complicating the spectrum. T o resolve multiple charge distribution envelopes, ultrahigh resolutions are r e q u i r e d (15). Unfortunately, commercial quadrupoles generally y i e l d only unit resolution throughout their mass range. Therefore, synthetic polymers that can typically contain a distribution of chain lengths and a variety of endgroups furnish quite a complicated mass spectrum, making interpretation nearly impossible. T h e approach we used to circumvent the difficulties described p r e viously are as follows. W e used sodium cations dissolved i n our mobile phase to facilitate ionization. T o simplify the resulting E S I spectra, we


4.

SIMONSICK & PROKAI

43

SEC with ESIMS Detection

reduced the number of components entering the i o n source. F u r t h e r more, the multiple charged states were reduced b y analyzing only small molecules that generally produce fewer charged states. Once we d e m onstrated that an electrospray signal was furnished i n a tetrahydrofuran ( T H F ) mobile phase, we evaluated the utility of S E C - E S I M S for S E C calibration, measurement of chemical composition distribution i n copolymers, and complex mixture analysis.

Experimental Details SEC was carried out using a three-column set of 1 0 , 500, and 100 Â 30 cm X 7.8 mm i.d. Ultrastyragel columns (Waters, Milford, M A ) . The acrylic macromonomers were analyzed using a two-column set of 500- and 100-Â Ultrastyragel columns. The T H F mobile phase was delivered by a Spectroflow 4000 solvent delivery system (Kratos Analytical, Manchester, U K ) at 1.0-mL/min flow rate. T H F is a flammable solvent and proper care should be exercised when using large volumes. A l l samples under study were dissolved i n the mobile phase ( ~ 0 . 5 % wt/vol) before analysis. The sample solutions were injected using a Rheodyne 7125 valve equipped with a 100uL loop (Cotai, CA). Effluent splitting was achieved with a T-junction (Valco) that supplied only ~ 8 - 1 0 min/flow to the mass spectrometer through a 25-cm-long fused silica capillary (25 μτη i.d.). A Spectroflow 757 absorbance detector (Kratos Analytical) operated at 254 nm was used for deter mining M W D data by SEC. The polystyrene calibrants used were molecular weights of 580, 2450, 5050, 11,600, and 22,000 D a with polydispersities ranging from 1.03 to 1.09. A block diagram of the S E C - E S I M S is seen in Figure 1. A Vestec 200ES instrument (Vestec Corp., Houston, T X ) was used to obtain the ESIMS (16). The spray was generated from the solvent entering


3

S E C - G P C Column(s) ι

ι

Injector

J- - - '

Flow Splitter

*

ESI-MS

U V or Rl Detector

Figure 1. studies.

Block diagram of the SEC-ESIMS instrumental setup used in all


44


the ion source through a 0.005 i.d. X 0.010 o.d. flat-tipped hypodermic needle held at 3.0 k V potential. Preformed ions were obtained by dissolving —5 X 10~ M sodium iodide in the T H F mobile phase. W h e n the needle tip to orifice distance was ~ 1 0 mm, the spray current was in the range of 6 0 - 1 0 0 μΑ at 5-10 ^ L / m i n flow rate. The source block was heated to 250 °C, and the spray chamber temperature was estimated at 5 5 - 6 0 °C. A Vec tor/One data system (Teknivent, St. Louis, M O ) was used to control the quadrupole analyzer (m/z 2 0 0 - 2 0 0 0 at 3 ms/Da scan speed). Selected-ion chromatograms were reconstructed from full-scan data, and elution volumes ( V E = t X flow rate) were determined by adjusting the time (t ) versus intensity data to the theoretical (Gaussian) elution (17) profile using a non linear curve-fitting program running on an IBM-PC/AT-compatible computer (MINSQ, Micromath Scientific Software, Salt Lake City, UT). The 80/20 (wt/wt) methyl methacrylate ( M M A ) rc-butyl acrylate (BA) macromonomer was prepared i n the following manner. To a 3000-mL flask 440.1 g M M A , 200.0 g ΒΑ, and 150.0 g methyl ethyl ketone (MEK) were added. The mixture was stirred and heated to reflux under a nitrogen blanket. After a 10-min hold, 30.0 g M E K , 0.140 g Vazo-67, and 0.050 g Co(dimethylglyoxime-BF ) were added to the flask. After a 5-min hold, 359.9 g M M A , 200.0 g M E K , and 1.90 g Vazo-67 were added over a 3.5h period. The mixture was held 1 h at reflux after the feed. Subsequently, 150.0 g M E K and 1.00 g Vazo-52 were feed over an hour. The mixture was held for 1 h at reflux. The mixture was then allowed to cool to room tem perature. A more detailed procedure and the Co(dimethylglyoxime-BF )2 synthesis are given in reference 18. 5


E

E

2

2

2

Results and

Discussion

Cationization has been the preferred technique for p r o d u c i n g gaseous ions from synthetic oligomers and polymers by desorption ionization (3, 4, 6,19). W e have relied on this approach upon considering the coupling of E S I to S E C . A small amount, ~ 1 0 ~ M , sodium iodide dissolved i n the T H F mobile phase does not impair the chromatography and affords meaningful E S I mass spectra as singly charged ions are seen as M [ N a ] , doubly charged as M [ 2 N a ] , and triply charged as M [ 3 N a ] . N o E S I M S signal was observed without addition of a soluble salt that p r o v i d e d the source of cations. T o simplify the complexity of the resulting E S I spectrum, we chose to reduce the number of components entering the E S I source. W e se lected S E C because the mode of separation is w e l l understood, p r e dictable, and performed on a routine basis i n our laboratory. T o reduce the breadth of the charge envelopes, we chose to examine exclusively lower molecular weight materials, typically < 3 0 0 0 D a . This molecular weight regime encompasses many of the components contained i n to day's high-performance coatings (19). W e have previously reported on the coupling of an S E C to a mass spectrometer operated i n the electrospray mode of ionization and its application to the molecular weight characterization of octylphenoxypoly(ethoxy)ethanol oligomers (20). T h e analysis of nonionic surfactants 5

+

+

+


4.


Rel.

600

tOO

45


SIMONSICK & PROKAI

1000

1200

1400

1000

1800

Int.

2000

m/z

Figure 2. Electrospray ionization mass spectrum of octylphenoxypoly(ethoxy)ethanol. Inset is the total ion chromatogram. Conditions are given in Experimental Details.

has also been accomplished by other condensed-phase separation tech niques w i t h mass spectrometric detection (21, 22). T h e following dis cussion serves as an introductory example of the data and its interpre tation. F o r a more detailed discussion on the coupling of S E C to E S I M S and its application to octylphenoxypoly(ethoxy)ethanol, consult refer ence 20. F i g u r e 2 shows the summed electrospray mass spectrum of an octylphenoxypoly(ethoxy)ethanol (see 1). T h e total ion chromatogram is seen i n the inset. T h e shaded region of the total ion chromatogram was summed to y i e l d the spectrum. T w o envelopes are present, due to singly and doubly charged species. T r i p l y charged ions are also present but are slightly masked by the doubly charged envelope. W e d i d not observe any other chemical species i n this surfactant other than 1 nor d i d we target our analysis toward such materials. Investigators have previously reported low-level impurities i n similar mixtures (23). T h e determination of charge state is easily accomplished by exam ining the repeat group. T h e molecular weight of the ethylene oxide unit ( - C H C H 0 - ) is 44 D a , hence the spacing i n the region above 1200 2

2

C

B

H

0CH CH

17

189

2

Da

2

j 4 4 n Da

OH + Na π

)| 40

Da |

1


46


D a . Close examination of the region from 600 to 1200 D a shows a 2 2 D a repeat unit due to the doubly charged oligomers. T h e base peak seen at 1549 D a is due to the sodiated η = 30 oligo mer. T h e octylphenyl [ C H - C H - ] moiety contributes 189 D a . T h e 30 ethylene oxide groups add 1320 D a , whereas the terminal h y d r o x y l group and sodium cation contribute 40 D a ; hence, the peak at [189 D a + 1320 D a + 40 Da] = 1549 D a . The equation describing the distribution of singly charged oligomers is 8

1 7

6

4

M [ N a ] = (229 + 44n) D a +

(1)


T h e doubly charged species follow the equation M[2Na]

2 +

= (126 + 22n) D a

(2)

L i k e w i s e , the triply charged species are described by the equation M[3Na]

3 +

= (91.67 + 14.67n) D a

(3)

T h e S E C elution behavior of any oligomer can be profiled by plotting the selected-ion chromatograms that correspond to the ions defined by equations 1-3. F o r example, the singly charged η = 20 oligomer fur nishes a singly charged ion at 1109 D a (see e q 1). T h e doubly charged η = 35 oligomer yields i o n at 896 D a (see e q 2). T h e triply charged η = 50 oligomer affords an ion at —825 D a as defined by equation 3. F i g u r e 3 displays the U V chromatogram (λ = 254 nm), the selected-ion plots of the singly charged η = 20 oligomer (1109 Da), the selected-ion plot of the doubly charged η = 35 oligomer (896 Da), and the triply charged η = 50 oligomer (825 Da) for the S E C analysis of octylphenoxypoly(ethoxy)ethanol (1). T h e fitted curves were generated using the nonlinear curve-fitting program described i n Experimental Details. As expected, the higher molecular weight η = 50 oligomer w i t h a larger hydrodynamic volume elutes before (19 min) the smaller η = 35 oligomer (20 min) and η = 20 oligomer (22 min). Such data can be used to calibrate the S E C and unrelated calibrants such as narrow molecular weight polystyrenes can be avoided (20). M W D information can be computed using the averaged mass spec trum presented i n F i g u r e 2. T h e doubly charged envelope w o u l d be used because a portion of the singly charged envelope exceeds the upper mass limit of our system. H o w e v e r , the reliability of this approach is poor due to instrumental parameters that may provide a nonuniform response w i t h molecular weight. M o r e o v e r , the electrospray response is not uniform w i t h increasing molecular weight. F o r example, the mo lecular weight average computed from the singly charged envelope is lower than that calculated from the doubly charged envelope. W e spec ulate that the multiple charging becomes predominant and attenuates


4.

SIMONSICK & PROKAI

47


2500

uv 2000

1500

1000


500

N

* η = 3 5 , [M + 2 N a f .•^,

; t;

π = 20, [M + N a f

^

η = 50, [M + 3 N a f

10

15

20

25

Elution Volume (mL)

Figure 3. UV chromatogram (λ = 254 mnj and selected-ion traces for octylphenoxypoly(ethoxy)ethanol. The triply charged η = 50 oligomer se lected-ion trace was obtained by summing 824-826 Da through the duration of the chromatogram. The doubly charged η = 35 oligomer selected-ion trace was furnished by summing 895-897 Da. The singly charged η = 20 oligomer selected-ion trace was obtained by summing 1108-1110 Da.

the relative abundance of the singly charged species proportionately to the increase i n molecular weight. W e recommend that selected i o n profiles be used for calibration. A full scan acquisition is collected and the selected-ion profiles of the individual oligomers used for calibration. H e n c e , the S E C is cali brated for octylphenoxypoly(ethoxy)ethanol using the octylphenoxypoly(ethoxy)ethanol oligomers. F i g u r e 4 presents the calibration curve obtained from the selected-ion plots of individual oligomers. Also plotted is the calibration curve obtained using narrow molecular weight p o l y styrenes. Notice the large discrepancy at lower molecular weights (

Size-Exclusion Chromatography with Electrospray Mass Spectrometric

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