Techniques for Liquid Chromatographic Columns Packed with Small Porous Particles Ronald E. Majors Varian lnsfrument Division, 2700 Mitchell Drive, Walnut Creek, Calif. 94598
A balanced density slurry technique was used to pack 5and 10-pm diameter porous particles into small diameter columns 15 to 50 cm in length. Results are presented for these columns operated in liquid-solid (LSC), liquid-liquid (LLC), and bonded-phase (BPC) modes of liquid chromatography (LC). In LSC, both silica gel and alumina gave columns which demonstrated good efficiency. In LLC, columns were coated by the in situ method. In BPC, silylation reactions were used to chemically bond a nonpolar and a polar phase to 10-pm silica. Multilinear solvent programming (gradient elution) techniques were used to separate wide range samples in LSC and BPC. Extension of separations obtained with thin layer chromatography and column LC with larger particles to small particles was made with much increased resolution per unit time. The small porous particles gave sharp peaks permitting better use of the refractive index detector and more sensitive detection with the ultraviolet detector. Separations of azo dyes, nitroaniline isomers, aromatic amines, plasticizers, and halogenated aromatic hydrocarbons are presented.
Recent advances (1-4) in preparation and packing of porous adsorbent particles with average diameters of less than 20 micrometers (pm) demonstrate theoretical predictions of increasing liquid chromatographic column efficiency with decreasing particle size. This improved efficiency results from increased rates of solute mass transfer into and out of small porous particles. Theoretical plate height, H , is proportional to d,1.8 where d p is the “effective” particle diameter for balanced-density slurry-packed silica gel in the particle size range of 5 to 40 pm (3). Performance of columns packed with small, porous particles now exceeds t h a t of the porous layer bead (PLB)adsorbents. In addition, the larger surface area per gram of totally porous particles allows larger samples to be injected without column overload. The present paper reports on the application and techniques for use of liquid chormatographic columns packed with porous particles of 10 wm or less. Separations using liquid-solid (LSC),liquid-liquid (LLC), and bonded phase (BPC)modes of LC are demonstrated.
EXPERIMENTAL Apparatus. The apparatus used to pack columns by the highpressure, balanced-density slurry procedure is reported elsewhere (2).
The liquid chromatography (LC) was performed using a Varian Aerograph 4200 Research Liquid Chromatograph with a Solventflow Programmer attachment. This chromatograph employs two constant volume rate delivery syringe pumps capable of output pressures up to 5000 psi. Injections were made on-column using the stop-flow technique through a high pressure, septumless injector (Varian Aerograph Part No. 02-001460-00). An Ultraviolet Absorption Detector and a Refractive Index Monitor, both available from Varian, were used to monitor column effluents. ( 1 ) J. F. K. Huber, Chimia, Suppl. 24 ( 1 9 7 0 ) . ( 2 ) R. E. Majors,Anal. Chem., 44, 1722 (1972). (3) J. J. Kirkland. J. Chromatogr. Sci., 1 0 , 593 (1972) (4) R. E. Majors, submitted for publication in J . Chrornatogr.Sci
Reagents. Soluents. Chromatographic solvents were of spectrophotometric grade available from Matheson, Coleman and Bell (Los Angeles, Calif.). A mixture of tetrabromoethane and tetrachloroethylene (J. T. Baker, Phillipsburg, K.J.) was used as the balanced density slurry solvent for silica and bonded-phase packings (2). For alumina, the higher density solvent, diiodomethane (Baker, density 3.3 g/ml), was used to suspend the small particles for high pressure slurry packing. Liquid phases, P,P’-oxydipropionitrile (ODPN) and Carbowax 400 (Union Carbide), were obtained from Varian. Solutes. Solutes were the best grade available and were used as received. Chlorinated insecticides (Kit No. 51 AX) were obtained form PolyScience Corp., P.O. Box 791, Evanston, Ill., 60204. Hindered phenolic antioxidants and stabilizers were provided, courtesy of the suppliers listed below: Name
Supplier
BHT (Butylated hydroxy toluene) BHA (Butylated hydroxy anisole)
Eastman Kodak Eastman Kodak
CAO-14 754 Goodrite Santowhite Powder
Catalin Corp. Ethyl Corp. B. F. Goodrich Monsanto
The sources and properties of the azo compounds are reported elsewhere (5). N-phenyl-beta-naphthylamine was purchased from K &K Laboratories, Plainview, N.Y. Other chemicals were obtained from the Organic “Chem Supply” Units (Chem Service Inc., 851 Lincoln Ave., West Chester, Pa.) Adsorbents. Silica gels were LiChrosorbs SI 60. They have an average pore diameter of 60 A and a surface area of 366 m2/g (3). LiChrosorb is available in 5-, lo-, 20-, 30- and 40-pm average particle diameters. Aluminas were LiChrosorb Alox T. They have surface areas of 70-90 m2/g and average diameters of 5 , lo-, and 30-pm. Samples of each were kindly provided by E. Merck (Darmstadt, W. Germany), their American affiliate, EM Laboratories, Inc., Elmsford, N.Y., or Varian Instrument Division, 611 Hansen Way, Palo Alto, Calif. 94303. MicroPak columns, prepacked with LiChrosorb SI 60, available in 1 5 , 25-, and 50-cm lengths, were obtained from Varian. Liquid phases were chemically-bonded to 10-pm LiChrosorb SI 60, as discussed under Procedures. Inquiries regarding the availability of adsorbents, bonded-phase packings, and prepacked columns similar to those herein described should be directed t o Varian. Thin layer chromatographic plates were E. Merck Pre-Coated TLC Sheets, Silica Gel F-254 and Aluminum Oxide F-254 Type T. They were purchased from VWR Scientific, P.O. Box 3200, Rincon Annex, San Francisco, Calif. Porasil C (Waters Associates, Framingham, Mass.) 30/60 mesh was used for the preparation of pre-columns in LLC experiments. Procedures. Column Preparation. The techniques used for packing columns by the balanced density slurry procedure are reported elsewhere (2). In LLC, columns were coated by the in situ technique, sometimes used to coat prepacked gas chromatographic (GC) columns (6). The liquid phase was dissolved in a volatile solvent, pumped through the column, and the solvent removed by passing dry helium through it. Directions for coating a prepacked silica gel column with approximately 30% ODPN are outlined below: A solution of 20% (v/v) ODPN in methylene chloride is passed through the column at a flow rate of 1-2 ml/min for one-half hour. The final coverage is dictated by the initial concentration of ODPN in the methylene chloride. ( 5 ) R. E . Majors and L. B. Rogers. Ana/. Chem., 41, 1058 (1969). (6) C. Horvath in “The Practice of Gas Chromatography.” L. S.
and A . Zlatkis, Ed., p 201
Interscience Publishers, New York, N . Y .
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 4 , A P R I L 1973
0
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755
Finish
Start
Table I. RF Values for Azo Dyes
Dye 1 2
3 4 5 6
Figure la. TLC separation of azo dyes
Plate: E. Merck Pre-coated TLC Sheet, silica gel F-254; solvent: 10% CH2Clp in hexane; development time: 50 min; solutes: shown on Figure
RF
0.69 0.39 0.21 0.098 0.064 0.015
lb
B) from separate pumps. The Solvent/flow Programmer Accessory permits the chromatograph to be used for either constant mobile phase composition for isocratic elution, solvent, or flow programming. Gradients were formed by continuous variation of the mobile phase composition at constant total pump displacement rates. Solvent programs were formed with the Multi-Linear Solvent Programmer (MLSP) which permits formation of both simple linear and complex nonlinear positive or negative gradients. Nonlinear programs may be generated by a series of one-to-ten linear steps of selectable slopes and duration. Column regeneration was accomplished by either negative retracing of the positive gradient or quick reset to the initial mobile phase composition.
RESULTS A N D D I S C U S S I O N Liquid.Solid Chromatography. Columns of silica and alumina are primarily for use in LSC. Silica is the most
I
0
I
1.2
I
I
1
I
2.4
3.6
40
6.0
Time ,min. Figure lb. LSC separation of azo dyes Coiumn: MicroPak Si-10; dimensions: 15 cm X 2.4 m m : mobile phase: 10% CH2C12 in hexane; flow rate: 132 m l / h r ; pressure: 350 psi; sample size: 1 pl; sample concentration: 0.2 mg/ml each; detector: UV
The column inlet is connected to a source of dry helium and is gradually pressurized from atmospheric pressure to 80 psi over the period of an hour. This operation permits slow removal of methylene chloride and leaves behind ODPN on the support. The ODPN is distributed evenly over the column by placing it in an oven at 75 "C for about an hour. A trickle (5-10 ml/min) of helium is passed through the column during this redistribution stage. To ensure mobile phase presaturation, a 20-cm by 6-mm precolumn was used containing 30% by weight of the respective liquid phase coated on 30/60 mesh Porasil C. Chemically-Bonded Phases. Liquid phases were bonded to 10-pm LiChrosorb SI 60 by reaction with organosilane reagents. A nonpolar phase of octadecylsilane groups and a polar phase of 0cyanoethylsilane groups were prepared. To eliminate residual surface hydroxyl groups, bonded-phase support materials were treated with trimethylchlorosilane (7). Bonded phase coverages were calculated from carbon-hydrogen content. The stable siloxane bonded phases resisted Sohxlet extraction for 24 hours each with benzene, acetone, and methanol in that order. Thin-Layer Chromatography. Prior to use, TLC plates were pre-equilibrated in a constant humidity environment. They were placed overnight in a desiccator containing a saturated solution of calcium chloride to give 31% relative humidity (8). After spotting, plates were developed in a chamber saturated with solvent vapors. All solutes were colored and were easily identified. RF values and H values were calculated from the plates (9). Column Chromatography. Mobile phases were custom-blended by automatic adjustment of the flow ratio of two solvents ( A and (7) M. F. Burke and R . K . Glipin, Anal. Chem. in press. ( 8 ) "The Merck Index." 7 t h ed., Merck &Co., Rahway, N . J . , 1960, p 1602. (9) S. G . Perry, R . Amos, and P. I. Brewer, "Practical Liquid Chromatography," Plenum Press, New York, N . Y . , 1972, p 9 .
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
general and widely used chromatographic adsorbent while alumina has selectivity advantages for certain classes of compounds. Adsorbent particles used in the present experiments are the same particles used on TLC plates only with narrower size distribution (3). For this reason, direct extrapolation from TLC to LSC can be made. Figure l a shows a TLC separation on a plate coated with Merck silica gel. Silica gel, having a slightly acidic surface, was better than alumina for the azo dye separation since those compounds are slightly basic. Six azo dyes were separated using 10% (v/v) methylene chloride in hexane. The development time was 50 min. The least polar, azobenzene, moved the fastest and the amino-substituted azo compound the slowest. The nitro-substituted dyes (No. 4 and 5 ) were unresolved. RF values for the dyes are given in Table I. Figure l b shows the corresponding LSC chromatogram on a 15-cm MicroPak Si-10 column using the same solvent system. As expected, the order of elution was similar to the TLC plate. All six compounds were separated in 5 min. In contrast to TLC, the nitro isomers (4 and 5 ) were separated to base line. Resolution per unit time increased for all peaks. They could be quantitated more easily than TLC by simple measurement of peak height (or area). For subsequent investigation, eluted compounds could have been collected individually a t the detector exit rather than by cumbersome removal from a TLC plate. Small diameter adsorbent particles possess the same adsorption characteristics as larger ones commonly used in column chromatography. Extension from classical column chromatography or LC on larger particles to small particle columns can be made with increased speed and efficiency. Recently, Randau and Bayer (IO) separated the isomers of nitroaniline on a 50-cm column of 30- to 40-pm dry-packed alumina Type T in about 8 min. Figure 2 shows base-line separation of the same in 45 sec on a 15-cm column of 10-pm slurry-packed alumina. Table I1 shows H, calculated from Figure 2, was a n order of magnitude lower for the shorter 10-pm alumina column even though linear velocity was 5 times greater than for the 50-cm larger particle column. Included in (10) D.
Randau and H . Bayer. J. Chromatogr., 66,382 (1972)
Table II. Comparative Efficiency and Speed for Nitroaniline Isomer Separation 30- to 40-pm Aluminaa
10-pm Aluminab
TLCC
Isomer
H. mm
N!td
H . mm
N .'t
H. mm
N,'t
o-Nitroaniline m-Nitroaniline p- N itroaniline
3.9 2.6 3.4
0.83 0.83 0.31
0.36 0.36
26 16 9.2
0.06 0.06 0.09
0.14 0.09 0.03
0.42
RF 0.22 0.14 0.09
From data of Randau and Bayer ( I O ) ;column: Aluminum Oxide Type T, 30- to 40-pm; column length, L = 500 mm; mobile phase, benzene linear velocity, v = 0.33 c m i s e c . Column: LiChrosorb Alox T. 10-pm; L = 150 mm, mobile phase, 40% ( v i v ) methylene chloride in hexane, v = 1.7 cm!sec. C Adsorbent: E. Merck Pre-Coated TLC Sheet, Aluminum Oxide F-254. Type T; development time = 31.5 min; v = 0.0044 c m / s e c . N ' t = plateslsec. a
4n4
TIME. MIN
t
"2
f
0.064ABS
I
0.016 ABS
NO,
I) 0-Nitroaniline
Z N 0 2
2) M-Nitroaniline
3) P-Nitroaniline
I
0
2
I
I
4
6
r 8
TIME, MIN
Figure 3. Solvent programmed separation of antioxidants on
5-pm alumina
I
0
Figure 2.
LSC
,
18 36 TIME,sec
54
separation of nitroaniline isomers on 10-pm alu-
mina Column: 10 p m LiChrosorb Alox T; dimensions: 15 cm X 2.4 mm; mobile phase: 40% CH2C12 in hexane; flow rate: 100 ml/hr; sample size: 1 pl; sample concentration: 1 mg/ml in methylene chloride; detector: UV
Table I1 are the RF values and H values of the three isomers on a n alumina TLC plate using the same solvent system. Efficiency in TLC surpassed the columns since the linear velocity was 2.6 X 10-3 times slower. However, Table I1 shows t h a t plates per second was the highest with 10-pm alumina, much lower with 30- to 40-pm alumina, and lowest with the TLC plate. Compounds best handled by LSC generally contain diverse functional groups (11, 12). Solvent programming (gradient elution) is often used in LSC for decreasing analysis time for samples which contain compounds with widely differing capacity factors, k'. Usually a n increase in mobile phase polarity is required to decrease k' values. Figure 3 shows a solvent-programmed separation of common hindered-phenolic antioxidants and stabilizers often used in mixtures for polyolefins in the 0.1 to 0.5 weight per cent range. A 15-cm LiChrosorb Alox T column containing 5-pm particles was used. A hexane-methylene chloride gradient was used to lower the k' of Santowhite Powder from approximately 100 (13) to 12. (11) L. R. Snyder in "Modern Practice of Liquid Chromatography," J. J. Kirkland. Ed., Wiley-lnterscience, New York, N.Y., 1971, p 143. (12) Ibid., p 206. (13) R. E. Majors in "Basic Liquid Chromatography," F. Baumann and N. Hadden, Ed., Varian Aerograph, Walnut Creek, Calif.. 1972, p 5-18.
Column: 5 p n LiChrosorb Alox T; dimensions: 15 cm X 2.4 mm; mobile phase: solvent A, hexane; solvent B, methylene chloride; flow rate: 60 ml/hr; sample size: 1 pl; sample concentration: 2 mg/ml of each;detector: UV; Samples 1. BHT, 2. CAO-14, 3. Triphenylphosphate, 4. Antioxidant 754. 5. BHA, 6. Goodrite, 7. Santowhite powder
Figure 3 includes a plot of the gradient slope used in the antioxidant separation. Column regeneration was accomplished by a negative retrace of the slope followed by a 5-min flush with the initial solvent composition. This procedure resulted in reproducible retention times. For peak 6 (Goodrite), a relative standard deviation in retention time of 11.8% or 209 1 3.8 sec was obtained for five replicate gradient runs. For peak 7 (Santowhite), a value of 3~2.6%was determined. An immediate reset to the initial mobile phase composition followed by sample injection resulted in very short, nonreproducible retention times for the low k' components. At least 30-45 min of flushing with hexane a t 1 ml/min was required for column regeneration and equally reproducible retention times. High performance LSC appears to be a very sensitive technique for quantitative analysis of antioxidants in polyolefins. Gas chromatography (14) and TLC (15) are used to determine antioxidants in polyethylene. In GC, column temperatures of 300 "C are required to elute common antioxidants from a silicone column (14). Because of broad peaks, for most the maximum sensitivity was about 1 fig using a flame ionization detector. I n Figure 3, the Santowhite peak represents an injection of 2 pg a t 0.16 Absorbance unit full scale. Assuming a minimum detectable absorbance of 2 X 1 0 - 4 Absorbance unit (16), about 10 ng of material might be detected. In TLC, 300-900 ppm of (14) J. A. Denning and J. A. Marshall, Analyst (London). 97, 710 (1972). (15) D. Simpsonand B. R. Currell. ibid., 96, 515 (1971). (16) M . N. Munk, J. Chrornatogr, Sci., 8, 481 (1970). A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 4 , APRIL 1973
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757
t
I
f
1.6X10-5RI Unit i
f
I
0
I
2
I
4
0.016 Abs
I
6
F i g u r e 4.
Separation of azo compounds using refractive index
Column: 10 p m Lichrosorb 3 6 0 ; dimensions: 45 c m X 2.2 mm; mobile phase: 5% CH2C12 in isooctane: flow rate: 60 ml/hr; sample size: 3 @ I containing 15 p g of compound 1 and 30 p g of compounds 2-4. Samples: 1. 4-(phenylazo)-N,N-diethyl-l-naphthylamine; 2 . N,N-diethyl-p-1-naphthylazoaniline; 3 . . N-ethyl-p-aminoazobenzene; 4. 3.5-diethyl-p-aminoazobenzene
I
I
2.4
0
TIME, min.
TIME, min
detector
I
4.8
Figure 5. Aromatic amine separation on ODPN-coated MicroPak Column: 32% ODPN on MicroPak Si-10; dimensions: 30 c m X 2.4 mm. mobile phase: isooctane: flow rate: 100 ml/hr: sample size: 1 PI; sample concentration: 0.4 p l / m l of N,N-diethyl- and N-ethylaniline; 1 mg/ml of others; detector: UV
T a b l e Ill. Liquid Phase C o v e r a g e as a
Function of
Initial Concentration
common phenolic antioxidants in polyethylene have been detected but not quantitatively determined (151 Because of their larger surface areas, small porous packings have greater sample capacity than PLBs. Larger samples required for less sensitive detectors can be injected without a sacrifice in column efficiency. Figure 4 shows a separation of azo compounds using the refractive index (RI) detector. Approximately 30 pg of each compound were injected. The narrow peaks give added response required by the RI. PLBs have limited utility with the RI since they must often be overloaded in order to observe minor components. High performance semi-preparative scale separations (10-100 mg) are possible using larger bore columns (27). Small porous packings are particularly attractive for larger columns required for prep LC because of their relatively low cost compared to PLBs. Liquid-Liquid Chromatography. In LLC, the balanced-density slurry packing procedure cannot be used easily with conventionally-coated packing materials because many liquid phases are soluble in the slurry solvents, For this reason, columns were coated by t h e in situ technique. Without prior standardization, it is difficult to accurately predict liquid phase coverage in the in situ method. Table Ill gives the weight per cent coverages of ODPN as a function of initial concentration in methylene chloride. The coverage (100 X x p / u s )of phase was determined by stripping the column with pure methylene chloride and weighing the liquid phase ( u p )after evaporation of the (17) D. R. Gere. C. David Carr, and S. Reifsneider, Eastern Anaiytical Symposium. New York, N.Y.. Oct. 1972.
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ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4 , A P R i L 1973
Initial concn ( v v % )
10
20 30 40
Coverage of ODPN (wt % ) a
16 33 45 59
f 2b f 2' f3 f 11
a Coverage caiculated as 100 X W, W , where W , is the weight of liquid phase and W , is the weight of silica gel in the column Each value is the average of at least two runs Average deviations are given For Carbowax 400 coverage was 17 f 2%
*
solvent. The weight of the silica ( b S )in the column was known from prior LSC columns. Each of t h e coverages u p to 45% were reproducible to about %IO%. The coverage of ODPN was difficult to control with initial concentration over 40% ( v / v ) since during the solvent removal step, droplets of excess ODPN appeared a t the column exit. The extent of coverage may differ with a different liquid phase. For example, Table I11 shows the coverage (17%) resulting from a solution of 20% (v/v) Carbowax 400 in methylene chloride. It was about half the coverage obtained for the same initial concentration of ODPN. Figure 5 presents a base- line separation of four aromatic amines on a 30-cm column of 32% ODPN on slurrypacked 10-pm silica using isooctane as a mobile phase. Elution order was the same as observed previously (18) on ODPN-coated Zipax (DuPont) and Corasil (Waters). Figure 6 shows t h e aromatic amine separation under the same chromatographic conditions but after removal of the ODPN and recoating the column with Carbowax 400. Note the increased selectivity of Carbowax relative to ODPN for the last two amines. Prepacked columns have (18) R. E. Majors, J. Chromatogr. Sci., 8 , 338 (1970).
FLOW SENSITIVE UV DETECTOR
NON-FLOW SENSITIVE UV DETECTOR
)DT
DT Ald
Aldri,
Mirez
f
T
0.016 Abs
I
T
Y
Mirf
Abs
lTDE
Dieldrin
,
9.6
TDE impurity 1
7.2
4.0 2.4 TIME , min.
0
Figure 6. Aromatic amine separation on Carbowax 400-coated M icroPak Conditions: same as for Figure 5 but column contains 17% Carbowax 400
i
0
I
uc I
2.4 4.0 Time, min
O
2.4Time, 4.0 min
7'2
I
7.2
Figure 8. Flow programmed separation of insecticides Aldrin
Conditions: same a s Figure 7 but flow rate was 30 mi/hr to 100 ml/hr at lO%/min. ( a j Flow sensitive UV detector; ( b ) Non-flow sensitive U V detector (79)
DDT
T 1
0.016 Abs
Methoxychlor
A 0
24
4.8
7.2
8.6 Time, min
12.0
14.4
16.8
Figure 7. LLC separation of insecticides Column: 33% ODPN on MicroPak: dimensions: 50 c m X 2.4 mm; mobile phase: isooctane; flow rate: 32 ml/hr; pressure: 375 psi; sample: 25-gg of each except 5 - p g of methoxychlor: detector: UV
been stripped and recoated repeatedly. Alternately, the liquid phase may be stripped and the column used for LSC. Figure 7 shows the separation of chlorinated insecticides on an in situ ODPN-coated 50-cm MicroPak column using isooctane as a mobile phase. At a 32 ml/hr flow rate, the
pressure drop was only 375 psi. Despite the relatively slow flow rate, methoxychlor (k' = 7.6) exhibited 2.3 effective plates/sec. With conventional LLC, flow programming is one of the few techniques used to decrease analysis time. By automatically flow programming, Figure 8a shows a threefold decrease in separation time for the insecticides while maintaining adequate resolution. For flow programming experiments, the Solvent/flow Programmer Accessory was used with a single pump. Its flow rate may be varied either linearly or nonlinearly as a function of time. Common to all commercially-available high performance UV detectors, a decreasing base line with increasing flow rate depicts flow sensitivity with organic solvents. By careful thermostating of the detector cell, flow sensitivity of the UV can be virtually eliminated (19) as Figure 8b shows. Bonded Phase Chromatography. In conventional LLC, mobile phase is in constant equilibrium with the coated liquid phase. This places certain requirements and restrictions on LLC: A presaturator column must be placed before the analytical column; column effluent is always contaminated with stationary phase making sample collection difficult; very high flow rates must be avoided, otherwise, liquid phase could be removed by shear forces; solvent programming is not feasible. By chemically-bonding the stationary phase to the support material, those problems are eliminated. Since both the mechanism and liquid phase characteristics may differ from conventional LLC, this form of chromatography (19) M Munk. Varian Aerograph, Walnut Creek Calif cation, 1972
,
private communi-
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4, APRIL 1973
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759
IA 'A
W
II
/
4.
O
N
=
OEt
1
5.
O
N
=
6.
O
N
=
N=
7. 12
O
Figure 9. Reverse phase matic hydrocarbons
24 36 TIME m ~ n
BPC
48
8.
separation of halogenated aro-
Column: octadecylsilane bonded to 10 p m LiChrosorb; dimensions: 25 c m X 2.4 mm; mobiie phase: solvent A , water; solvent B, isopropanol; temperature: 50 "C; flow rate: 100 ml/hr; sample size: 6 PI; sample concentration: 1 m g / m l each except 5 m g / m l of hexachlorobenzene in ethanol; detector: UV 11
O MeOC-
a 12.
N
=
IMPURITIES
Figure 11. Structures of azo dyes used in Figures 12-15
D PHTH
DECYL BENZYL
0
I
I
6
TIME. min
Figure 10. BPC separation of phthalate plasticizers on "cyano" phase Column: "Cyano" phase bonded to 10 p m LiChrosorb: dimensions: 15 c m X 2.1 mm; mobile phase; 5 % CH2CI2 in hexane; flow rate: 123 ml/hr; sample size: 1-pl, sample concentration: 0.4 p l / m l of didecyl and decyl benzyl phthalate and 1 mg/ml of dibenzylphthalate in isooctane; detector: UV
. 0
2
4
6
8
IO
12
14
16
I8
TIME, MIN
might be termed "Bonded Phase Chromatography," or BPC, to differentiate it from the latter. Two popular types of bonded phases have found use in BPC. Silicate esters are prepared by direct esterification of alcohols; with surface hydroxyls of silica (20). T h e Si0 - C bond is undesirable for BPC because of its hydrolytic and thermal unstability. Siloxane bonds are prepared by silylation of surface hydroxyls (21-23). The Si-0-Si bonds (201 I. Halasz and I. Sebastian, Agnew. Chem.. Inf. Ed. Engl., 8, 453 (1969), (21) W A . Aue and C. R. Hastings, J , Chromatogr., 42, 319 (1969). (22) J J. Kirkiand and J. J . DeStafano, J. Chrornatogr. Sci., 8, 309 (1970). (23) J. J. Kirkland. J. Chrornatogr. Sci., 9, 206 (1971).
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Figure 12. Solvent-programmed separation of azo dyes on silica gel Column: MicroPak Si-10; dimensions: 15 c m X 2.1 mm; mobile phase: solvent A , hexane containing 0.2% isopropanol; solvent 8, methylene chloride containing 0.2% isopropanol; solvent program: shown on chromatogram; flow rate: 60 ml/hr; sample size: 4 pl; sample concentration: 0.3 mg/ml each except 0.09 mg/ml of 9 and 10
are stable and siloxane liquid phases can be used at elevated temperatures with aqueous or alcoholic solutions (24). (24) J. A . Schmit, R . A . Henry, R. C. Williams and J. F. Dieckman, ibid, 9 , 645 (1971).
“,%;:If 20
Carbon-hydrogen analysis revealed 5.1 0.3% (average deviation) by weight of phase, based on a (OSi(
