Anal. Chem. 1987, 59, 327-333 (4) Hanaoka, Y.; Murayama, T.; Muramoto, S.; Matsuura, T.; Nanba, A. J. ChrOmetOgr. 1982, 239, 537-548. (5) Davis, J. C.; Peterson, D. P. Anal. Chem. 1985, 57, 768-771. (8) Hwang, H.; Dasgupta, P. K. Anal. Chem. 1988, 58, 1521-1524. (7) Haginaka, J.; Yasuda, H.; Uno, T.; Nakagawa, T. Chem. fharm. Bull. 1983, 31, 4436-4447. ( 8 ) Haginaka, J.; Yasuda, H.; Uno, T.; Nakagawa, T. Chem. fharm. Bull. $984, 32, 2752-2758.
327
(9) Haginaka, J.; Wakai, J.; Yasuda, H.; Uno, T.; Nakagawa, T. J. Llq. ChrOMtGgr. 1985, 8 , 2521-2534. (10) Haginaka, J.; Wakai, J.; Yasuda, H. Chem. fharm. Bull. 1986, 3 4 , 1850-1852.
for review
9, 19g6* Accepted September 16,
1986.
Ultrasonic Micronebulizer Interface for High-Performance Liquid Chromatography with Flame Photometric Detection J. F. Karnicky* and L. T. Zitelli Varian Research Center, 611 Hansen Way, Palo Alto, California 94303
Sj. van der Wal* Varian Instrument Group, 2700 Mitchell Drive, Walnut Creek, California 94598
An ultrasonic micronebuilrer Interface for effective nebuliration at 2-20 pL/min liquid was developed to allow the determination of nonvolatlle analytes by mlcrobore HPLC wlth virtually unmodified gas chromatographic detectors. A detectablllty of 50 pg/s phosphorus was obtalned wlth a dynamlc range of 3 decades and acceptable peak broadening using a flame photometrlc detector (FPD) as a detector. Solvent compatlblllty appears mainly determined by the choice of chromatographlc detector. Dual-wavelength operatlon can improve the signal-to-noise ratio by a factor of 5, improve the preclslon from 6 to 2 % , and eliminate base-line shifts. The utility of the micronebulirer-FPD as a selective HPLC detector Is demonstrated in the analysis of phospholipids and sugar phosphates.
Detectability with the flame photometric detector (FPD) (1) in gas chromatography is approximately 1 pg/s phosphorus. With the thermionic specific detector (TSD) (2) detectability is better than 0.1 pg/s phosphorus and 0.1 pg/s nitrogen. These excellent mass detectabilities together with the P and N selectivity make the FPD and TSD attractive detectors for micro-HPLC. Unfortunately, direct introduction of flow rates of even a few microliters of liquid per minute into an unmodified gas chromatographic detector is restricted to volatile mobile phases and solutes ( 3 , 4 ) .Even for these volatile mobile phases and solutes differential volatilization of solutes and solvents leads to a narrow range of operating conditions. Too little volatilization (short residence time or low temperature in the direct interface tube) results in a noisy base line when the (first) flame of the detector receives a high load of mobile phase; too much volatilization causes precipitation of less volatile components in the interface tube, noticeable as spiking, and eventually clogging of the tube. An interface for effective nebulization at 2-20 pL/min liquid was therefore developed. Initial attempts to use concentric or cross-flow pneumatic nebulization failed to yield reliable results for truly nonvolatile solutes, i.e., solutes that cannot be analyzed by gas chromatography in an underivatized form, cf. ref 5. Cross-flow nebulization on a sintered disk (6) was not successful below
Table I. Apparatus component
model
manufacturer
conditions
HPLC
5560
Varian
split flow, 2-20
injector rf signal generator
7520 185
Rheodyne
1-pL injection swept from 3.25 to 3.45 MHz
rf amplifier
240L
E.N.I.
power meter
43
Bird
pL/min
(2)
Wavetek
at 10 Hz continuous wave, 7-15-W output
frequency 5381A Counter thermocouple 151-786 temperature controller liquid N2 D-6000 Dewar FPD (1) K-filter S-10-767-F K-PMT R910 gas flowmeters 602/60l
Cole Parmer
modified
Varian Corion Hamamatsu Matheson
dual-beam mode
hydrogen air
liquid air liquid air
105, 175
helium (for iniector)
liquid air
90 psi
Hewlett-Packard Cole Parmer
-114 "C to -70 O C
105-175 mL/min 135 mL/min mL/min
20 pL/min aqueous mobile phase, which precludes its use with an unmodified FPD or TSD. An alternative to developing a low flow rate nebulizing interface would be adaptation of the detector to the standard HPLC flow rates (ca.1mL/min). This is naturally much more costly. it has been applied to the FPD by Julin et al. (7) and Chester (8) and has resulted in detectabilities of more than 1 ng/s phosphorus, which renders it less attractive. The objective of this paper is to report on the nebulizer interface we have developed and its utility in allowing the use of an FPD as a micro-HPLC detector.
EXPERIMENTAL SECTION A summary of the main equipment components is given in Table I. A Model 5560 high-pressure liquid chromatograph
0003-2700/87/0359-0327$01.50/00 1987 American Chemical Society
328
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2. JANUARY 1987
. mu"*.
'I
I
uniform distribution of droplets with diameters less than 10 r m is formed. These are swept out of the nebulizing chamber and transported to the FPD by the air supply to the first flame (air 1). The nonnehulized fraction of eluent is wicked away from the excitation point and drained from the nebulizing chamber. The combination of high-frequency focused excitation, precise positioning of the needle relative to the diaphragm, and efficient wicking of the nebulizing chamber results in the minimal peak broadening needed to operate at 2-20 rL/min HPLC flow rates. The ultrasonic transducer is operated in a frequency-swept mode in order to achieve smooth, stable nebulizatim. Water is circulated through the nebulizer hody in order to cool the transducer crystal and provide a medium for focusing the ultrasonic waves. Forward rf power to the transducer is typically on the order of 5-15 W reflected power is typically 3-10 W. In the transport tube connecting the nebulizer to the FPD, the droplets of the aerosol desolvate, i.e., volatile mobile-phase components evaporate from the droplets 80 that the aerasol particles now consist of the nonvolatile mobile-phasecomponents, sample, and residual volatile material suspended in a vapor consisting of the air 1 carrier gas plus evaporated solvent. It is desirable to prevent as much of this evaporated solvent as possible from entering the FPD to avoid overloading of the flames. This is accomplished by a condensation technique analogous to that used by Dickinson and Fassel (10) in their ultrasonic nebulizer ICP system. The aerosol passes through a condenser which is ccoled to a temperature just above the solvent's freezing point. Most of the evaporated solvent is condensed to a liquid and drained from the transport tube. Some (possibly 50%) of the particles containing the sample are also lost in this process. The remaining particles (containing the sample and nonvolatile mobile-phase components) are swept into the FPD along with residual solvent by the noncondensable air 1 flow. Temperature control is achieved by varying the rate a t which nitrogen gas is boiled from a reservoir. In the modified FPD (Varian, details in Figwe IC) the particles pass through the first flame into the second (analytical) flame. This second flame is in a normal (noninverted) configuration. Phosphorus in the sample is converted to HPO, the emission of which is observed by a PMT through the appropriate filter. Simultaneously, a potassium emission signal from a nonvolatile potassium salt that is added to the mobile phase is observed at 180O to the phosphorus detection. Not shown in Figure 1are interlocks which, for example, shut off power to the nebulizer if the cooling water is not circulating. All solvents were obtained from Burdick & Jackson (Muskegon, MI); the samples were purchased from Sigma (St. Louis, MO). Extra-column hand broadening was determined by a method analogous to that of Kok et al. (11). The peak widths for a series of nucleotide monophosphates eluting a t differing values of k' were measured under nonoverloading conditions in an ion-pairing reverse-phase system. Equal theoretical plate heights were assumed for all the peaks, and the measured peak variances were extrapolated to zero retention time to obtain the extra-column contribution.
RESULTS AND DISCUSSION Aam, A*, 1
w
e 1. Schema& of miaonebullzer-FPD detector. Fw details see Experimental Section. (Varian, Walnut Creek, CA) was used in the split-flow mode throughout this study. The columns were made of untreated 0.32-mm4.d. fusedsilica t u b w or 1-mm-i.d. stainless-steel tubing, packed with Micropak SP C18-3, Micropak SP Protein C18-5, or Micropak SP Si-3 material (Varian) a t Varian. A schematic of the micronebulizer-FPD interface is shown in Figure 1. Details of the micronehulizer (9)and of the modified FPD are shown in parts B and C, respectively, of Figure 1. HPLC effluent is introduced through a 70-mm X 0.005-in.-i.d. stainless-steel tube onto the surface of a ca. 0.030-in.-thick glass diaphragm which is vibrating at high frequency due to the focused ultrasomc excitation produced hy a piezaeledic crystal A fraction of the eluent (10-70% depending on mobile-phase composition, flow rate, and rf power) is nebulized at the excitation point. A
Performance of the Nebulizer. Nehulizer stahility was initially determined by measuring the attenuation (by light scattering) of a laser beam passing through the aerosol at the exit of the nebulizer while nebulizing water. Subsequently, the stability of the combined nehulizertransport tube was measwed by using the FPD to monitor the phosphorus signal from continuous nebulization of a dilute phosphate solute in water. Both methods indicated a short-term coefficient of variation of about 8% in the nebulizer output with a 1-s detedor time constant. This variation visibly correlated with pulsations of aerosol production at the excitation point within the nebulizer body. This 8% coefficient of variation is about a factor of 3 larger than the variability of the ultrasonic nebulizers in atomic absorption or inductively coupled plasma which operate a t much larger gas and elemental analysis (E), liquid flow rates. Essentially, our nebulizer is not able to benefit from the averaging effect of a large mixing volume such
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
329
Table 11. Solvents Used for Nebulizer-FPD %
cosolvent
%
Rapg/s P
isooctane
90
10 10 20
-103
90 80 50 50 90 50
2-propanol 2-propanol 2-propanol methyl tert-butyl ether tetrahydrofuran dioxane dioxane
190
isooctane
650 620
50
700
-88 -88 -88 -114
solvent
isooctane isooctane
isooctane cyclohexane cyclohexane toluene methyl tert-butyl ether tetrahydrofuran tetrahydrofuran dioxane
acetone 2-propanol 2-propanol
methanol water
50
260
10
450 400 e 950 1000 480
50
100 100 100 90
methanol
10
100 100 100 100 100 100
T,b"C
RTd RT -88
RT -102 -87
test compdc L L L L L L L L L L
S
L,s L
e
RT
700
280
-89 -88
5000
RT
130
-88
S S S
50
RT
UMP
D = mass flow rate of phosphorus giving twice the peak-to-peak noise. T = temperature in the condenser. "L = lecithin, S = spingomyelin, UMP = uridine monophosphate. d R T = room temperature (18-23 "C). eNo signal.
as is present in these systems because one of the design specifications is extra-column band broadening on the order of 1-2 pL. Thus, nebulizer noise limits the overall detectability and peak quantitation of the system. The effect of this detector noise is considerably reduced by the double-beaming technique to be discussed below. Performance of the Nebulizer-FPD. The factors involved in nebulization, transport, and FPD detection by our system were most thoroughly characterized for solutes in water. The FPD when used as a gas chromatographic detedor is known to be a mass flow rate detector, with emission proportional to the amount per time unit of phosphorus entering the flame (1). We measured FPD emission spectra while introducing a variety of nebulized solutions into the flame. These measurements indicated that the flame processes for the nebulized nonvolatiles in water were the same as for the gas-phase samples (Le., HPO emission). In none of the above studies did we see any indication of reduction of this response (quenching) due to the presence of water or any clear indication of concentration effects. The detectability of the nebulizer-FPD for nonvolatile analytes in water was 50 pg/s phosphorus compared to a value of 2 pg/s that was measured for this FPD under identical operating conditions when used as a GC detector. The difference is due to a &fold increase in base-line noise due to the nebulizer and a &fold reduction in the sensitivity (measured as the detector current divided by the mass flow rate of phosphorus into the nebulizer or, equivalently, out of the column). Because of the very low liquid flow rates involved, we were not able to determine quantitatively how much of this reduction in sensitivity is due to (a) nebulization inefficiency, (b) transport inefficiency, and/or (c) decreased FPD response to nebulized material vs. gaseous material. Qualitative observations of the amounts of water condensed in the transport tube and drained from the nebulizer are consistent with the assumption that this factor of 0.2 represents nebulizer and transport inefficiency, with the nebulization step itself accounting for most of the loss. This efficiency of transport. and nebulization is consistent with results reported for the use of ultrasonic nebulizers and desolvation tubes in inductively coupled plasma atomic emission spectroscopy (10, 13, 14). The corollary conclusion is that the flame response to nebulized material is essentially the same as that for gaseous material. This is made possible by the extremely small droplets generated by ultrasonic nebulization. We have measured these droplets (by differential laser light scattering) to be 4 pm in diameter. Considering 2 pg of nonvolatile compound
eluting as a 1-min peak at 20 pL/min, one can readily calculate that the diameter of the desolvated particle which enters the FPD is about 0.2 pm. This is presumably sufficiently small to allow complete reaction of the sample. Mobile-Phase Compatibility. Employing an FPD as a detector does not allow the use of acetonitrile or halogenated solvents. Acetonitrile gives a large response of its own due to the formation of CN radicals. Halogenated solvents form halo acids that corrode the FPD. Toluene and dioxane are attractive for selectivity and room-temperature operation of the condenser, respectively, but there was no phosphorus response in these solvents. Results of experiments nebulizing organic solvents into the FPD with the ultrasonic micronebulizer are shown in Table 11. When characterizing nebulization systems it is important to use nonvolatile test compounds like lecithin and sphingomyelin to be able to discriminate between volatilization and nebulization. For nonpolar solvents a cosolvent was needed to dissolve the lecithin and sphingomyelin. No correlation was observed between any single physical parameter and the signal-to-noise ratio for different solvents. Limiting the hydrocarbon flux to the first flame of the FPD by lowering the temperature of the desolvation chamber to its lowest permissible value (cf. Table 11,2-propanol; isooctane 2-propanol) is clearly beneficial. Flow rates of organic solvents were 2-8 pL/min, which is optimal for the 0.32-mm X 150-mm 3-pm particle size fused silica columns as well as for the micronebulizer FPD. Optimal performance of the FPD, when nebulizing organic mobile phases, is achieved by increasing the ratio of air to hydrogen in flame 1. This results in a significantly larger signal (and approximately 2 times the noise) than that seen with the standard, more fuel-rich, flame conditions. This is attributed to a more complete combustion of quenching organic molecules to CO or C 0 2 in flame 1 (hence less chemical interference in the analytical flame). The detectability values of 200-1000 pg/s phosphorus in organic mobile phases are worse by a factor of 100-500 than those for the FPD used as a gas chromatographic detector without quenching interferences. Base-line noise while nebulizing organic eluents ranged from 5 times the gas chromatography value (e.g., methanol) to ca. 50 times (e.g., tetrahydrofuran). Sensitivity of the nebulizer-FPD with some organic mobile phases (e.g., ethers) is significantly less than with alcohols or aqueous eluents. We were not able to quantitatively attribute the reduction in sensitivity to nebulizer efficiency, transport efficiency, and reduced flame response. However, visual observation of ample
+
330
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
nebulization and the large improvement in response that occurred with solvent condensation indicate that the largest part of the reduced sensitivity in organic mobile phases is due to quenching of the HPO emission by the presence of large amounts of organic material in the flame. This phenomenon is well-known in use of the FPD as a GC detector and has been observed ( I , 2,15,16)in other nebulization-FPD experiments (7,17). The detectability of 50 pg/s phosphorus in water warrants a further investigation of the present nebulizer-FPD system, since more than 60% of current HPLC separations are performed on reverse-phase columns with aqueous mobile phases (18).
Dual-Wavelength Operation. When buffer salts, ionpairing reagents, or other additives are present in the aqueous mobile phase, the fluctuations in nebulizing efficiency introduce noise into the FPD base-line signal that limits detectability, and dual-wavelength operation becomes very important. These fluctuations in nebulizer efficiency also give the chromatographic bands a ragged appearance near the peak apex and increase the coefficient of variation of the peak area. The effect of these fluctuations on the coefficient of variation of the peak area is significant to the extent that the frequency of the fluctuations matches the frequency components of the chromatographic peak. We have measured the frequency distribution of this nebulizer noise and found that a t low frequencies it is dominated by l / f noise and that the frequency distribution corresponds to a chromatographic peak with half-width on the order of 1 s. In order to minimize the effects of this nebulizer noise, we have developed the following techniques for referenced emission measurements. The emission signal at the analyte wavelength is written as
i
Consider first the case where the Ksm and KRMiare small (no significant increase in emission due to the mobile phase). In this case, eq 4 becomes
(5) Further, if t,(t) and eR(t) are equal (or proportional), which we expect to be approximately true for a nonvolatile analyte and nonvolatile reference material, the signal in the referenced chromatogram will be proportional to the concentration of phosphorus in the mobile phase. In this case, peak quantitation, but not detectability, will be improved by dualwavelength operation. Consider next the case where the mobile-phase components contribute significantly to the emission background. In this case, if the efficiencies of nebulization and transport for the analyte, reference material, and emitting mobile-phase components are equal, eq 4 becomes x K S M i ci i
= CKRMiCi i
S ( t )is the PMT anode current (nA) measuring flame emission at the analyte wavelength As (e.g., 526 nm for HPO detection). So is the flame background (nA) a t As and is assumed to be constant. F is the volume flow rate (pL/min) of mobile phase entering the nebulizer. KsMi is the response of the FPD a t As to the mass flow rate into the detector of the ith mobilephase component (nA/(ng/s)). ci is the (constant) concentration in the mobile phase of the ith component (ng/pL). q(t) is the (dimensionless) efficiency of nebulization and transport of component i. Ksp is the response of the FPD at As to phosphorus entering the detector (nA/(ng/s)). t,(t) is the efficiency of nebulization and transport for the phosphoruscontaining analyte. c,(t) is the concentration of phosphorus in the mobile phase as it enters the nebulizer. Note that although concentration terms appear in eq 1, the flame is responding to the product F-c,(t), which is a mass flow rate with units of ng/s. Assume that there is another component in the mobile phase (not one of those represented in eq 1)that emits light in a spectral region where the analyte does not and does not emit light a t As. The emission signal a t this reference wavelength will be
R ( t ) = Ro
+ CKRM~
tj(t)
Fc,+ KR
tR(t)
FCR
(2)
1
R(t)is the PMT anode current (nA)measuring flame emission a t the reference wavelength, XR. Ro is the flame background (nA) a t XR, and is assumed to be constant. The K R ~are i the response factors for the mobile-phase components a t XR (nA/(ng/s)). tR(t) is the efficiency of nebulization and transport for the reference material. cR is the (constant) reference material concentration in the mobile phase. We define the referenced emission signal as
+
KSP
+ KRCR Ci K R M ~+ CK R~c R
c,(t)
(6)
In this case, the first term manifests itself as the base line, and the peaks in S&) are proportional to the concentration of analyte. Detectability and peak quantitation will both be improved by dual-beam operation. (If the various efficiencies in eq 4 were unequal but proportional, eq 6 would differ but the conclusion would not.) Therefore, we expect dual-wavelength operation to improve detectability to the degree that the various efficiencies exhibit the same temporal behavior. For components of low volatility we expect good compensation. This is borne out by our experimental results. A series of experiments were conducted evaluating various spectral features for use as the reference signal. Emission spectra were measured from 250 to 870 nm while nebulizing a variety of mobile phases into the flame. Promising spectral features were than tested as reference wavelengths. We found that the best results were obtained by adding a potassium salt in a concentration of a few millimolar to the mobile phase and using the K+ emission at 767 nm as a reference signal. If eq 6 held exactly, noise in the referenced signal due to variations in the nebulizer efficiency would disappear. In practice, we found an improvement in signal-to-noise ( S I N ) ratio up to a factor of 5 as illustrated in Figure 2. The coefficient of variation of the peak area for CMP in 1 mM KOH was reduced from 6% to 2%. These improvements are an encouraging validation of the assumptions underlying eq 6.
Besides improving S I N ratio, dual-wavelength operation can be used to reduce gradient base-line shift. Two examples are given in Figures 3 and 4. In Figure 3, the increasing ammonium acetate concentration causes the base-line signal and base-line noise a t 526 nm to increase. By contrast, the ratioed trace shows little drift. Note, however, that the noise reduction at the end of the gradient relative to the beginning is illusory as the ratio a t constant signal decreases automat-
331
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
0.0
0.0
3.0
6.0
9 .o
12.0
15.0
9.0
12.0
15.0
Minutes
4.0
8.0
12.0
16.0
20.0
Minutes Flgure 4. Dual wavelength with base-line noise reduction and constant sensitivity: as in Figure 3, except for mobile-phase compositionsmobile phases (A) 10 mM ammonium acetate, 3.1 mM potassium acetate, and 0.15 mM dipotassium hydrogen phosphate and (B) 200 mM ammonium acetate and 3.0 mM potassium acetate.
t
A526
0.0
3.0
6.0
Minutes Flgure 2. Illustration of the effect of dual-beam operation: column, 170 X 0.32 mm Micropak SP IP-5; mobile phase, 1% n-butyl alcohol, 0.06% tetrabutyiammonium hydroxide, 0.12% acetic acid, 0.008% perchloric acid; pressure, 304 atm; split flow operation; sample, 0.2 fig each of CMP, 5-AMP, %AMP, 2-AMP, and CAMP, in order of elution.
1
5
10
20
50
F (plhin)
Flgure 5. Measured extra-cdumn variance using the micronebulizer-FFQ detector. Units equal s2. For details see Experimental Section.
~~
0.0
4.0
8.0
12.0
16.0
20.0
Minutes Flgure 3. Gradient base-line correction by double-beam operation: column, 150 X 0.32 mm Micropak SP IP-5; mobile phases (A) 5 mM ammonium acetate and 0.57 mM potassium acetate and (6)200 mM ammonium acetate and 5 mM potassium acetate in H,O; split flow operation, 0.4 mL/min via shunt, ca. 5 fiL/min via column; pressure, 110 atm; linear gradient, 0 100% B in 10 min; curve I, 526 nm; curve 11, 526 nm/767 nm.
-
ically 10-fold with an increase of the reference ( K ) signal by a factor of 10. Another implementation of the dual-wavelength technique is shown in Figure 4 where a phosphate salt is added to the starting mobile phase and the potassium acetate concentration at the end of the gradient is kept low, resulting in a steady but noisy 526- and 767-nm signal and a 526 nm/767 nm ratio trace with constant sensitivity over the whole range of the gradient. It was found that at higher salt concentration the 526- and 767-nm signals do not track as well as at lower concentrations, causing the base-line noise in the ratio to increase from 2%
(peak-to-peak) at 1-10 mM to 6 7 % at 100-200 mM salt. This is presumably due to instability of the flame at high salt concentrationsin the mobile phase. Completely involatile salts cause a spiking signal and precipitate past the first flame, and thus, only ammonium salts could be used in high concentrations. No problems were encountered during several weeks of use of 0.1-1 mM potassium acetate, which is the optimum concentration for double-beam operation. The NH,+/K+ selectivity of the reverse-phase column causes an even larger change in the 526 nm/767 nm trace in gradient elution if the gradient is started at less than 10 mM NH,+ salt. The base-line shift caused by cation exchange may be improved by using a lower concentration of a stronger anion (e.g., citrate) and a higher concentration of potassium salt. Dynamic Performance of the System. Memory effects were negligible: less than the carry-over due to injection. Extracolumn band broadening of the system as a function of flow rate is shown in Figure 5. For a compound with a retention time of 20 min and a column with 5000 theoretical plates the extra-column variance contributes less than 10% to the total peak broadening, if the flow rate is at least 2 fiL/min. The less than 10% criterion is often used as an indicator for acceptability.
332
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987 IO0
3
;ens
IO
B
40t 30 1
1
1
ngPis
-
10
4 oa
Figure 6. (a) Linearity of the response to CMP in 0.1 M ammonium acetate: (0)peak height (nA), (A)sensitivity (PA per ng of PIS). (b) Linearity of the response to UMP in 5 mM dipotassium hydrogen double beam ((A52e/A767) phthalate: (0)single beam (526 nm), (0,A) X A 7e7). (c) Sensitivity as a function of phosphorous flux in the detector: (0)single beam, (A)dual-beam corrected;same data as Flgure 6b.
Linearity of the response to mass flow rate of phosphorus was measured in aqueous eluents (cf. Figure 6). The dynamic range does not exceed 3 decades. The lower limit depends on the base-line noise level in the particular eluent. The upper limit is determined by the onset of self-absorption of HPO in the flame of the FPD ( I ) .
As mentioned before we can only speculate about the causes of the observed nonlinearity of the response. One possible reason for the nonlinearity at low mass flow rates of the analyte is a nonlinear quenching effect by a mobile-phase component. At high mass flow rates the decrease in sensitivity is likely due to changing flame chemistries as in gas chromatographic operation of the FPD ( I ) . The capacity of the cooling apparatus for the desolvation chamber did not allow for continuous operation for more than 2 h. Sensitivity and noise level were measured repeatedly over the 6-month period that one of the nebulizer-FPD systems was employed as a microbore HPLC detector. The sensitivity was reproduced between 2-h operation periods with a 5% coefficient of variation, while the noise level was increased after 6 months operation by a factor of 3 due to occasional use with mobile phases containing high concentrations of nonvolatile buffer components like ammonium perchlorate. Applications. The phospholipids are a group of polar lipids that are prevalent in nature. An excellent review of phospholipid (PL) analyses was recently published (29).The lack of a good, inexpensive detector is the main obstacle for PL analysis. Detection by means of a flame ionization detector with a moving-belt interface (20) or infrared absorption detection at 5.75 or 6.15 pm (21)suffers from lack of sensitivity. Postcolumn reactor detection schemes are developed for specific detection of phospholipids and triglycerides (22) but are complex. Conductivity (23) and refractive index (24) detectors are not very selective. UV absorbance detection at 195-203 nm (24,25)has often not enough selectivity for crude extracts and has no uniform response to phospholipids. The more sensitive two-stage postcolumn reactor for phosphorus is specific but complex and hard to miniaturize and has not yet been demonstrated for phospholipids (26). The most sensitive and selective, but unfortunately also the most expensive, detector used with PL is the mass spectrometer with a moving-belt (27) or thermospray interface (28). From the detection limits of the micronebulizer-FPD one can calculate a minimum detectable quantity of 300 pmol for a 1-min PL band in methanol, which is 10 times worse than for SIM-MS with a moving-belt interface (27) and does not offer an improved SIN ratio over UV absorbance detection at 200 nm. The latter detection method, however, allows only the analysis of standards and extensively purified extracts, while the micronebulizer-FPD may be applied in the analysis of crude extracts because of its superior selectivity. Most phase systems used for P L separations have a mobile phase containing chloroform, dichloromethane (20, 27), or acetonitrile (25,29,30)and are not compatible with the FPD detector. Gradient elution with a compatible mobile phase (31)using a silica column was not successful and would result in high detection limits. We chromatographed a bovine brain extract on a Micropak Protein CI8 column in an aqueous methanolic mobile phase and compared it with UV absorbance detection of the crude extract and of a purified sphingomyelin fraction at 205 nm (Figure 7 ) . Although the experimental column was not very efficient, the differences in selectivity are clearly visible: most glycolipids elute before the main phospholipid components. The dependence of molar absorptivity at 200 nm on the degree of conjugation of the sphingomyelin component could be used for discrimination when a UV absorbance and a (selective) nebulizer-FPD detector are used in series as with the UV and nonselective refractive index detector (32). Another important group of compounds that have no distinct chromophores and are hard to separate and detect are the sugar phosphates. As with other organophosphates (26)
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
333
phosphate is shown in the highly selective (but low-efficiency) phase system of Figure 8.
ACKNOWLEDGMENT We thank Dr. Robello for his prototype electronic amplifiers, M. Cole for his technical assistance, and M. Bobinecz and M. Antonini for their help in preparation of the manuscript.
LITERATURE CITED 10.8 14.4 18.0 Minutes Flgure 7. Analysis of lipid extract with UV absorbance (A, B) and micronebuiizer (C) detection: column, 150 X 0.32 mm Micropak 98% methanol in water, 10-min Protein Cle; mobile phase, 90 gradient, spilt flow; pressure 280-240 atm. (A, C) Methanolic supernatant of 40 g of Type V I 1 bovine brain extract (1:2 chloroform/ methanol). (B) Bovine brain sphingomyelin, 10 g, methanolic supernatant. 0.0
3.6
7.2
-
k
0.0
I 3.0
hc,
I
I
I
6.0
9.0
12.0
15.0
Minutes
Flgure 8. Example of sugar phosphate analysis by HPLC wlth micronebulizer-FPD detection: column, 250 X 0.32 mm Mlcropak SP IP-5; mobile phase, 10 mM octylarnine, 0.5 mM potassium perchlorate, 0.1 mM ammonium hydrogen phosphate, 0.5 mM potassium acetate, pH 5; sample, 1 wg of glucose-6-phosphate and 1.5 p g of glucose-1phosphate (in order of elution).
it was found necessary to add a phosphate salt to the mobile phase to diminish peak broadening. A higher concentration of phosphate impairs sensitivity and selectivity, so a compromise has to be made between efficiency and selectivity. A rapid separation of glucose-1-phosphate and glucose-6-
Patterson, P. L.; Howe, R. L.; Abu-Shumays, A. Anal. Chem. 1978, 5 0 . 339-344. Patterson, P. L. J. Chromatogr. 1978, 167, 381-397. McGuffln, V. L.; Novotny, M. V. J. Chromatogr. 1981, 218, 179-187. Gluckman, J. C.; Novotny, M. J. Chromatogr. 1984, 314, 103-110. Plotczyk, L. L.; Larson, P. J. Chromatogr. 1983, 5 4 , 211-226. Layman, L. R.; Lichte, F. E. Anal. Chem. 1982, 5 4 , 638-642. Julin, B. 0.; Vandenborn, H. W.; Kirkland, J. J. J. Chromatogr. 1975, 112, 443-453. Chester, T. L. Anal. Chem. 1980, 5 2 , 1621-1824. Karnicky, J. F.; Zltelll, L. T. U S . Patent 4582654, April 15, 1986. Dlckinson, G. W.; Fassel, V. A. Anal. Chem. 1989, 41, 1021-1024. Kok, W. Th.; Brinkman, U. A. Th.; Frei, R. W.; Hanekamp, H. B.; Nooitgedacht, F.; Poppe, H. J. Chromatogr. 1982, 237, 357-369. Sturman, B. Varlan Techtron Pty. Limited, personal communication, M a y 26, 1983. Berrnan, S. S.;McLaren, J. W.; Willie, S. N. Anal. Chem. 1980, 5 2 , 488-492. Taylor, C. E.; Floyd, T. L. Appl. Spectrosc. 1981, 35, 408. Aldous, K. M.; Dagnall, R. M.; West, T. S Analyst (London) 1970, 95, 417. Veillon, C.; Park, J. Y. Anal. Chim. Acta 1972, 6 0 , 293. Chester, T. L. Anal. Chem. 1980, 5 2 , 638-642. Majors, R. E. LC Mag. 1984, 2 , 660-668. Aitzetmuller, K. Fette, Seifen, Anstrlchm. 1984, 8 6 , 318-322. Kiuchi, K.; Ohta, T.; Ebine, H. J. Chromatogr. 1977, 133, 226-230. Chen, S. S.-H; Kou, A. Y. J. Chromatogr. 1984, 307, 261-269. Compton, B. J.; Purdy, W. C. Anal. Chim. Acta 1982, 142, 13-29. Bouyoukos, S. A.; Armentrout, D. N. J. Chrornatogr. 1980, 189, 61-71. Compton, B. J.; Purdy, W. C. Anal. Lett. 1984, 17, 1857-1862. Jungalwala, F. B.; Evans, J. E.; McCluer, R. H. Biochem. J. 1975, 745, 517-526. Priebe, S.R.; Howell, J. A. J . Chromatogr. 1985, 324, 53-63. Jungalwala, F. B.; Evans, J. E.; McCluer, R. H. J. LipldRes. 1984, 2 5 , 738-749. Kim, H-Y.; Salem, N., Jr. Anal. Chem. 1988, 5 8 , 9-14. Chen, S. S.-H.; Kou, A. Y. J. Chromatogr. 1982, 227, 25-31. Hsieh. J. Y-K.; Welch, D. K.; Turcotte, J. G. Lipids 1981, 76, 761-763. Hax, S. M. A.; Geurts van kessel, W. S. M. J. Chromatogr. 1977, 142, 735-741. Compton, B. J.; Purdy, W. C. Anal. Lett. 1984, 17, 1857-1862.
RECEIVED for review February 28,1986. Accepted August 18, 1986.
