Internal surface reversed-phase silica supports for liquid

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Anal. Chem. 1905, 57, 1757-1763

Internal Surface Reversed-Phase Silica Supports for Liquid Chromatography I. Helene Hagestam and Thomas C. Pinkerton* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

A new concept In llquld chromatographic packlng materlai Is described. The new supporls are designed with glycerylpropyl bonded external surfaces in order to be nonadsorptlve to protelns and with polypeptlde phases, for the partltlonlng of hydrophobic analytes, bound only to the internal surfaces of porous sllica. The nominal pore dlameters are kept small to exclude protelns from the Internal surface region. The new packing supports enable the HPLC analysls of hydrophobic drugs in serum or plasma by dlrect lnjectlon. Large-partlculate internal surface reversed-phase precolumns, utlllred In conjunction wlth a C18 enrichment column and a C18 analytical column, facilitate the quantlflcation of phenytoin In plasma by direct lnjectlon over a concentration range from 6 to 24 pg/mL wlth an average coefficient of variation of 2.5 %. Small-partlcuiate hlgh-performance Internal surface reversed-phase anaiytlcal columns demonstrate the HPLC Separation of an anticonvulsant drug mixture In serum by direct Injection, wlthout adverse column fouling due to protein accumulation.

One of the most significant problems currently facing the analysis of drugs in serum or plasma by HPLC is the timeconsuming sample pretreatment prior to chromatographic injection. Since many drugs are small, nonvolatile hydrophobic organic molecules, reversed-phase liquid chromatography has become the most widely employed analytical technique for the separation and quantification of such analytes in serum or plasma. Protein containing matrices, such as serum and plasma, can not be directly injected onto reversed-phase C18 HPLC columns, because proteins denature a t the reversed-phase interface, adsorb onto the support particulates, and accumulate inside the chromatographic column. An initial accumulation of proteins within an HPLC column deteriorates column performance by clogging particulate pores, thus inhibiting the diffusional mass transport of analytes into the porous packing and decreasing column capacity ( I ) . Excess accumulation of proteins within a column decreases the interparticulate space and constrains the flow of the mobile phase. In order to avoid destruction of small particulate, reversed-phase HPLC columns, various sample cleanup schemes have been developed (2-8). Each involves the separation and isolation of analytes from the protein matrix. The most classical pretreatment method encompasses the removal of proteins by precipitation, followed by organic extraction of the analytes from the supernatant, evaporation of the extraction solvent, and rec6nstitution of the analyte residue in the mobile phase, before injection onto an HPLC column (2). Disadvantages of this conventional serum sample cleanup procedure are coprecipitation of analytes with proteins, loss of analytes during extraction, and time consumption. Advantages of this procedure include removal of endogenous species and preconcentration of the analytes. A second method of serum sample cleanup involves the use of off-line solid phase extraction of analytes onto disposable, large particulate

silica bonded phase supports (3). Although this technique can be automated, serum proteins remain on the packing, thus only one disposable column can be used per sample. A third method of serum sample cleanup incorporates large particulate (30-50 pm diameter) silica bonded reversed-phase (4-7) or ion-exchange materials (8), packed into short precolumns and connected in-line with HPLC columns by switching valves. In most cases, with this latter method, the analytes are strongly retained on the precolumns along with the proteinaeous substances. The analytes a e subsequently back flushed from the precolumns with strong solvents onto HPLC columns. Purge washes of the precolumns are generally required to remove residual amounts of retained proteins. Periodic replacement of the precolumns is required after a hundred or so injections. Although this precolumn technique has the advantage of preconcentrating the analytes, it suffers from the time-consuming interruptive back flush, purge washes, and precolumn replacement. The following research demonstrates a novel chromatographic reversed-phase support, termed internal surface reversed-phase (ISRP) packings, which accommodates the direct injection of serum samples without the destructive accurnulation of proteins. The fundamental principle behind the internal surface reversed-phase concept is to confine the hydrophobic partitioning phase exclusively to the internal particulate region of a porous silica support, while keeping the external surface of the support hydrophilic and nonadsorptive to proteins (Figure 1). By producing the ISRP supports with small pore diameters, all proteinaceous substances can be excluded and recovered in the column void volume, while the small hydrophobic analytes penetrate the particulates and interact with the internal hydrophobic partitioning phase. In this work, the new internal surface reversed-phase concept is demonstrated either though the on-line isolation of the drug phenytoin from human plasma with a large-particulate, low-performance ISRP precolumn, followed by separation of the drug fraction on a conventional C18 HPLC column or though the direct isolation of phenytoin from plasma and concomitant separation from other anticonvulsant drugs on a small-particulate, high-performance ISRP column.

THEORY The internal surface reversed-phase supports represent a new concept in liquid chromatography, which combines the fundamental principles of size exclusion and bonded phase partitioning separations to produce a type of surface discriminating chromatography. An expanded form of the general retention equation is

Vr = VO+ KsecVp + Kp(i)Vs(i)+ Kp(e)Va(e)

(1)

where VI is the solute retention volume, V , is the interstitial volume between the packed particulates (i.e., the “external void volume”), K,,, is the solute size exclusion coefficient, V,, is the penetration volume, and are the solute partition coefficients, relative to the internal and external surface phases, and Vs(i)and Vs(e) are the internal and external partitioning phase volumes, respectively Clearly, if solutes can

0003-2700/85/0357-1757$01.50/0@ lg85 American Chemical Society

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and ionic strength to prevent protein precipitation (12). In principle, if a small pore diameter chromatographic silica support can be produced with an external hydrophilic glycerylpropyl bonded surface and an internal hydrophobic partitioning surface, then solute retention volumes, with respect to each size discriminated solute class, would diffnr significantly.

EXPERIMENTAL SECTION ISRP Support Derivatization. The production of the ISRP supports is facilitated by first modifying small pore diameter silica with a glycerylpropyl bonded phase. To this phase is covalently bound polypeptides with hydrophobic moities which are susceptible to enzyme cleavage. The derivatized packing is then treated with enzymes to remove the hydrophobic species from only the external surface owing to the fact that the selected enzymes are too large to gain access to the internal surface region

Flgure 1. Cutaway view of internal surface reversed-phase support particulate: (A) protein, (B) analyte, (C) hydrophilic giycerylporpyl bonded external phase, (D) hydrobobic polypeptide Internal partitioning phase.

be confined to interacting with either the external or the internal surface, discriminating capacity factors can be achieved from modifying the surfaces with different partitioning phases. For the dynamic isolation of proteins from small molecular analytes in blood plasma, the macromolecular proteins can be confined to the external region of a silica particulate by selecting small pore diameters. Among the primary plasma proteins (serum albumins, globulins, and fibrinogen), the human serum albumin is the smallest and present in the greatest abundance. The serum albumin has a molecular weight of 65 600, a prolate ellipsoid shape, with dimensions of 150 8, X 38 A, and a hydrodynamic radius of gyration R, of 31.0 A, as measured by light scattering (9). In estimating the size exclusion limit, serum albumin may be treated by the “solid sphere” model yielding a “solid sphere” radius r of approximately 40 8, (Le., R, = 0.78r) (IO). Since spherical silica particulates are composed of agglomerated silica microspheres, a square cross sectional pore model can be invoked ( 1 0 , and the ratio of solute radius to effective pore radius of 1.00 would yield complete size exclusion (i.e., K, = 0) (IO). This implies that the pore diameter of the silica particulates must be less the 80 8, in order to exclude human serum albumin from the internal region of the porous silica. Typically the pore diameter of reversed-phase silica supporta ranges from 50 to 70 8, for packing material used in the separation of small molecular species. The total specific surface area for these supports ranges from 150 to 400 m2/g. Since the greatest amount of surface exists within the intraparticulate space, small analytes partition predominantly on the internal surfaces of a support packing. In contrast, the plasma proteins cannot gain access to the internal surface region and only interact with the external surface. On reversed-phase C18 supports protein adsorption results from the nonspecific, multisite hydrophobic partitioning which leads to denaturation. In contrast, protein adsorption is minimal on glycerylpropyl bonded silica (i.e., “diol” phases), when a mobile phase composition is maintained at an appropriate pH

(13). The low-performance ISRP packing material is produced from Glycophase GPC 40 (Pierce), a 37-74 pm particle diameter glycerylpropyl bonded “controlled pore glass” with a nominal pore diameter of 40 A. The glycerylpropyl bonded phase silica is activated with carbonyldiimidazole (CDI) as previously described (14). The resulting silica is then derivatized with either the dipeptide, glycine-L-phenylalanine(Gly-L-Phe)or the tripeptide, glycine-L-phenylalanine-L-phenylalanine (Gly-L-Phe-L-Phe). The hydrophobic phenylalanine moieties are then cleaved from the external surface of the support particulateswith carboxypeptidase A. The amount of polypeptide present on the particulate surface, before and after enzyme treatment, is determined by subjecting the material to acid hydrolysis and subsequently quantifying the released phenylalanine by conventional reversed-phase HPLC, with fixed wavelength detection at 254 nm. The high-performance ISRP packing material was produced from Hypersil (Shandon), a 5-pm underivatized silica. The silica is modified with a glycerylpropyl phase (15),and then derivatized with polypeptides in the same manner as described above, with subsequent cleavage of the phenylalanine moieties from the external surface with carboxypeptidase A. A more detailed description of the ISRP preparation process is given elsewhere (13). Chromatographic Apparatus. The low-performance ISRP packings are evaluated in a precolumn extraction mode. The chromatographic system (Figure 2) consists of the ISRP precolumn (A), a short C18 enrichment column (B), and a C18 analytical column (C). P1 and P2 are single piston Milton Roy Model 396 pumps (Laboratory Data Control). Linear pneumatic pulse dampeners are connected in series with each pump. Pump P1 delivers an aqueous 0.1 M phosphate/0.2 M sodium sulfate buffer (pH 6.0), at a flow rate of 1.5 mL/min, through a low-pressure system to a glass precolumn (A), 10 cm X 3 mm i.d., containing the large particulate ISRP material. A Brownlee 40-pm C18 reversed-phase cartidge column, 3 cm X 4.6 mm i.d. (not shown in Figure 2), is placed in-line with pump P1 to remove trace organics from buffer S1. Pump P2 delivers an isocratic mobile phase of 20% acetonitrile/33% methanol/47% 0.1 M phosphate buffer (pH 6.0), at a flow rate of 1.0 mL/min, through a highpressure system consisting of column B, a 3 cm X 4.6 mm i.d. stainless steel cartidge column containing 10-pm C18 reversedphase packing (Brownlee), and column C, a 25 cm X 4.6 mm i.d. reversed-phase HPLC column, containing prepacked 5-pm U1trasphere support (Altex). Columns A, B, and C are connected by valve V2, a six-port stainless steel Rheodyne Model 7010 switching valve. Serum and plasma samples are introduced by means of 30-pL Teflon loop on V1,a low-pressure Tefzel injector valve (Altex). D1 and D2 are fixed wavelength Beckman Model 153 ultraviolet-visible detectors equipped with 254-nm filters. The high-performance ISRP chromatographic system consists of a single piston Milton Roy Model 396 pump, a linear pneumatic pulse dampener, a Rheodyne Model 7010 injector valve with a 20-pL loop, a high-performance ISRP column, and a fixed wavelength Beckman Model 153 ultraviolet-visible detector equipped with a 254-nm filter. The high-performance ISRP supports, made from 5-pm Hypersil, are slurry packed into a 25 cm X 4 mm i.d. stainless steel columns with a constant volumne pump. The eluent for the high-performanceISRP system consists

ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985

_I

i-l

D1

1

Figure 2. Liquid chromatographic system employed for phenytoin quantiflcation with ISRP precolumn extractions: (A) ISRP precoiumn, (e) C18 enrichment column, (C) C18 analytical column, (D1 and D2) 254-nm ultraviolet detectors, (L) sample injector loop, (PI & P2) single piston pumps, (S1 and S2) solvent containers, (V1 and V2) six-port switching valves, (W) waste container.

of 20% acetonitrile/80% 0.1 M phosphate buffer (pH 6.0). ChromatographicProcedure. To test the dynamic extraction of analytes from a plasma sample onto the large particulate ISRP supports, a plasma sample containing phenytoin is introduced to the ISRP precolumn (A) by means of injector V1 with the aqueous buffer S1. The proteins are eluted in the void volume of the ISRP precolumn through valve V2 (dashed-line position, Figure 2) to detector D1. After elution of the proteins, valve V2 is switched (solid-lineposition, Figure 2), and the phenytoin which possesses a moderate capacity factor on the ISRP column, is shunted onto the C18 enrichment column B. Switching valve V2 is then returned to its original state (dashed-he position) enabling the drug fraction to be eluted onto the C18 high-performance column C for further separation, with quantification by detector D2. Reagents and Standards. Water used in the preparation of chromatographic mobile phases was distilled, then circulated though a mixed ion-exchange bed, activated charcoal bed, and 0.2-rm filter incorporated within a Gelman Water I purification unit. Mobile phase methanol and acetonitrile was HPLC grade (Fisher). The potassium dihydrogen phosphate used in preparing the aqueous buffers was HPLC grade (Fuher). AU organic solvents and mobile phase buffers were filtered prior to use with 0.22-pm Nylon-66 membranes (Millipore). The polypeptides and bovine carboxypeptidase A were obtained from Sigma Chemical Co. Standard human serum albumin was obtained from Miles Laboratories, and pooled human plasma, containing heparin, was obtained from a local blood bank. The phenytoin (i.e., 5,5-diphenylhydantoin) used in preparation of standard solutions for quantification was 99+% grade from Aldrich Chemical Co. The anticonvulsant standards (phenytoin, phenobarbital, and carbamazepine), used in the high-performance ISRP separation, were acquired in lyophilized serum from Fisher Diagnostics.

RESULTS AND DISCUSSION Production of ISRP Supports. The ISRP packing material is produced from commercially available controlled pore silica particulate supports. The silica is covalently modified with a glycerylpropyl bonded phase to render the surface nonadsorptive to proteins. Hydrophobic polypeptide partitioning moieties, containing chemical bonds which are sus-

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ceptible to enzyme cleavage, are covalently bound to the glycerylpropyl groups on the silica surface. This derivatization is carried out with molecules that can penetrate the porous particulates, so both the internal surface as well as the external surface of the silica possess the hydrophobic partitioning phase. Since the hydrophobic molecules on the external surface are adsorptive to proteins, the packing is treated with an enzyme which cleaves off the hydrophobic moieties from the external surface. The internal surface is not affected by the enzyme treatment owing to the inability of large enzymes to enter the small pores of the silica. In principle, a variety of polypeptides, including any combination of amino acids containing hydrophobic groups, can be used to produce the hydrophobic internal surface partitioning phase (13). In this work, two polypeptides were selected, a dipeptide, glycine-L-phenylalanine (Gly-L-Phe) and a tripeptide, glycine-L-phenylalanine-L-phenylalanine (GlyL-Phe-L-Phe). Each of these moieties produced partitioning phases with the solute retention properties equivalent to a conventional phenyl phase support. The enzyme selected to remove the hydrophobic moieties from the external surface was carboxypeptidase A, an exopeptidase which cleaves terminal L-amino acids sequentially with specificity for the free carboxyl group (16). Although the carboxypeptidase favors aromatic amino acids, it also nonspecifically cleaves a variety of hydrophobic aliphatic amino acids. Since the volume of a spherical macomolecule is proportional to the third power of its molecular weight (IO), the radius of gyration for carboxypeptidase A (molecular weight of 35000) is estimated to be approximately 24 A, implying a ”solid sphere” radius of about 31 A. This means the pore diameter of a silica support must be less than 60 8, to confine the enzyme to the external surface. One must keep in mind, however, that silica supports with nominal pore diameters of 40-60 A may have wide pore diameter ranges; thus penetration into the larger pores might be expected. The carboxypeptidase can be confined to the “external” surface, but since the enzyme is smaller than the serum proteins, the enzyme will have greater surface access than the serum proteins. This means that, once the “external” hydrophobic groups have been cleaved with the carboxypeptidase A, it is unlikely that the remaining hydrophobic moieties would be accessible to the larger serum proteins. Large Particulate ISRP Supports for Precolumn Extractions. The ISRP supports were first produced with large particulate silica supports and demonstrated in a low-performance precolumn extraction mode for reasons of cost containment. The low-performance ISRP supports were produced from CPG40 Glycophase, a large particulate (37-74 pm diameter) glycerylpropyl bonded silica with a nominal pore diameter of 40 A. The initial surface coverage of the glycerylpropyl bonded phase was determined to be 397 rmollg, by means of a paraperiodic acid oxidation assay described elsewhere (17,18). Neither the human plasma proteins nor the phenytoin analyte interacted with the glycerylpropyl bonded phase, as illustrated by the elution and complete recovery of the proteinaeous substances in the column void (Figure 3A). The hydrophobic partitioning phase coverages of the large particular ISRP peptide supports are given in Table I, along with performance data. After enzyme treatment, the G~Y-LPhe dipeptide support (CPG40-gph) yielded a 96 rmol/g internal peptide phase, while the Gly-L-Phe-L-Phe tripeptide support (CPG40-gph2)yielded a 47 pmol/g internal peptide phase. The elution of human plasma from the tripeptide modified CPG40 Glycophase, which had not been enzyme treated, is illustrated in Figure 3B. The plasma proteins are seen to interact with the external hydrophobic phenylalanine

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plasma b.1 A.U. 10.1 A.U.

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0

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7 0 . 0 0 2 A.U.

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0

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Figure 3. Chromatographic elution of human plasma and phenytoin from large particulate precolumns: (A) underlvatlzed CPG40 Glycophase,

(e) tripeptide derivatlzed CPG40 Glycophase (nonenzyme treated), (C) ISRP tripeptide CPG40 Glycophase (enzyme treated); mobile phase, 0.1 M phosphate/0.2 M sodium sulfate (pH 6); flow rate, 1.5 mL/mln; pressure, 150 psi; detector D1,254 nm; sample size, 30 pL.

Table I. Phase Coverage and Performance Evaluation of Large Particulate CPGlO Glycophase Peptide Modified Supports

partioning phase

enzyme cleavage

dipeptide CPG40-gph

untreated enzyme treated (ISRP) untreated enzyme treated (ISRP)

tripeptide CPG40-gphz

ISRP Droduction phase coverage," rmol/g % phase removed 166 96

42

64 47

27

.,

anaiyte retention k6 N/mc 9.6 8.4

I

.

180

190

protein eluti-u11 % recovery of plasma proteinsd 97 100

19.2

120

88

12.1

190

108

Partitioning phase coverage in micromoles of peptide phase per gram of support. Capacity factor ( k ') of phenytoin on packed columns (3 mm i.d. X 10 cm). 'Efficiency of column, relative to phenytoin, in plates per meter. dPercent recovery of human plasma proteins on the modified packing material, relative to the underivatized CPG4O Glycophase support, on first injection of plasma sample with an aqueous mobile phase of 0.1 M orthophosphate/0.2 M Na2S04,pH 6 (flow rate 1.5 mL/min). phase, as indicated by a lower recovery (Table I) and the extreme chromatographic tailing of the protein peak, which obscures the elution of the analyte. The elution of the human plasma from the enzyme treated tripeptide modified CPG40 Glycophase (Le., the ISRP column) is illustrated in Figure 3C. The removal of the phenylalanine moieties from the external surface eliminates the interaction of the plasma proteins with this support, resulting in a clean elution of the proteins, and an approximate 100% recovery, compared to the underivatized CPG40 Glycophase. The phenytoin analyte successfully partitions with the peptide phases of the ISRP precolumns, yielding capacity factors given in Table I. As expected, the tripeptide phase had a higher capacity factor than the dipeptide phase, and the non-enzyme-treated supports had a slightly higher capacity

factor than the enzyme-treated material (Table I). This demonstrates that the capacity factor of the analyte can be increased by the addition of phenylalanine moieties. This means, in principle, one could design an almost unlimitless number of internal surface partioning phases with the desired selectivity and strength, simply by varying type and length of the peptide phase. Surprisingly, the enzyme treated supports also exhibited a greater efficiency than the nonenzyme-treated packing (Table I). This is attributed to widening of pore entrances by the removal of phenylalanine moieties from regions immediately adjacent to the pores on the ''external" surface, thus facilitating better diffusional mass transport of the analyte with the internal stationary phase. Quantification of Phenytoin in Plasma by HPLC with ISRP Precolumn Extration. The retention of the phenytoin

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Table 11. Quantification of Phenytoin in Human Plasma by HPLC Analysis with Extraction on Tripeptide ISRP Precolumn concn. of phenytoin added to plasma

mean concn

coeff of

measured"

variation, %

6 10 15 20 24

6.03 f 0.11 10.02 f 0.22 14.87 f 0.62 20.07 f 0.51 25.05 f 0.48

2.2 4.2 2.6

1.9

1.9

@Meanf standard deviation, with three to four replicate measurements.

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Flgure 4. Chromatographic elution of phenytoin from C18 analytical

column, after extraction from plasma with ISRP tripeptlde precolumn. Points A, 6,and C are described in the text. The conditions were as follows: mobile phase, 20% acetonitrile/33% methanol/47% 0.1 M phosphate (pH 6); flow rate, 1.O mL/min; detector D2, 254 nm; sample, 30 pL of 24 pg/mL phenytoin dissolved in human plasma. on the ISRP tripeptide support was sufficient for precolumn switching isolation of the phenytoin from the plasma proteins. The chromatographic system utilized for this analysis is illustrated in Figure 2. A typical chromatographic elution, as recorded by detector D2 at the outlet of the C18 analytical column C, is illustrated in Figure 4. At point A the plasma sample, spiked with phenytoin, is injected onto the ISRP precolumn. An aqueous buffer transverses the ISRP precolumn carrying the plasma proteins to waste, while the phenytoin migrates slowly down the ISRP precolumn. After about 2 min (point B), valve V1 is switched, and the phenytoin is shunted onto the C18 enrichment column B. Collection of the phenytoin onto the enrichment column continues for about 5 min, after which valve V2 is switched (point C) and the analyte fraction is eluted onto the C18 analytical column C, by means of a 20% acetronitrile/33% methanol/47% 0.1 M phosphate mobile phase. The phenytoin is seen to elute from the analytical column along with unidentified impurities (Figure 4). The retention of the phenytoin, under these mobile phase conditions, is sufficient to accommodate the resolution of other anticonvulsant drugs or metabolites commonly observed in the HPLC analysis of phenytoin in human plasma (19-21).

A stock solution of phenytoin in methanol was diluted with human plasma to produce concentrations covering the normal phenytoin therapeutic range (6-24 pg/mL). By use of the chromatographic procedure described above, a calibration curve was developed over this concentration range with phenytoin dissoved in methanol. The subsequent quantifi-

cation of phenytoin in plasma was accomplished by the same chromatographic procedure and reference to this standard curve. The recovery of phenytoin from the human plasma ranged from 97 to 100% of the total phenytoin added, by comparison to the phenytoin dissolved in methanol. Since it is known that the phenytoin undergoes protein binding with serum albumin in excess of 90% (22,23),the protein bound phenytoin, apparently, is released from the protein on introduction to the ISRP column. Quantification of free phenytoin in the plasma, by an ultrafiltration method described elsewhere (23),on dilution with the chromatographic eluent, indicates that the phenytoin is not displaced from the albumin by constituents of the mobile phase. The HPLC assay of the phenytoin in plasma with the ISRP precolumn extraction technique, therefore, yields total phenytoin concentration, over this concentration range, with an average coefficient of variation of 2.5% (Table 11). The efficiency of the analytical column remain constant with the performance of 100 phenytoin assays, indicating that no residual proteinaeous substance ever reached the highperformance chromatographic system. The ISRP tripeptide precolumn withstood in excess of several hundred plasma injections over a period of approximately 3 months without any indication of deterioration. Neither the ISRP precolumn or analytical column ever required purging with strong solvents or back flushing. The unique feature of the ISRP supports is that the proteins are completely flushed though the precolumns. The ISRP precolumns, therefore, provide a valuable alternative to existing sample cleanup procedures when assaying hydrophobic drugs in plasma. The ISRP precolumns function to dynamically extract the drug analytes from a plasma matrix, thus protecting conventional reversed-phase analytical columns from proteinaceous contaminants. Small Particulate ISRP Supports for High-Performance Separations. A high-performance ISRP column was produced to evaluate the viability to utilizing the internal surface reversed-phase concept as a means of conducting high-performance analyte separations on serum samples by direct injection. A tripeptide glycine-L-phenylalanine-Lphenylalanine ISRP column was produced from 5 pm diameter Hypersil silica. The silica was derivatized with a glycerylpropyl bonded phase and the tripeptide was attached to the glycerylpropyl groups in the same manner as described above. As with the low-performance material, the phenylalanine moieties were removed from the "external" surface by enzyme cleavage with carboxypeptidase A. The resulting high-performance ISRP tripeptide material was packed into a stainless steel analytical column (25 cm X 4.6 mm i.d.) and connected, in the standard fashion, between a stainless steel loop injector and a 254-nm fixed wavelength detector. Onto the ISRP tripeptide analytical column was injected 20-pL samples of human serum containing various anticonvulsant drugs. The serum proteins eluted in the void volume with a 94% recovery. The protein fraction was immediately followed by unidentified

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,

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5

10

15

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10

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(MIN)

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Flgure 5. Chromatographic Separation of anticonvulsant drugs in human serum by direct injection onto the high-performance ISRP trlpeptlde column: (1) human serum proteins and endogenous small molecules, (2) phenobarbltal, (3) carbamazeplne, (4) phenytoin; mobile phase, 20% acetonitrlle/80% 0.1 M phosphate (pH 6); flow rate, 1.0 mL/min; detection, 254 nm; sample size, 20 pL.

hydrophilic small molecules and the anticonvulsant drugs phenobarbital, carbamazepine, and phenytoin, respectively (Figure 5). The three hydrophobic drugs were well resolved, and all components were eluted in under 12 min, with a 20% acetonitrile/80% 0.1 M phosphate buffer mobile phase. The quantitative recovery of phenytoin from the human serum samples was 98%, indicating that the assay with the highperformance ISRP column also yielded the total drug concentration. Interestingly, the elution order of carbamazepine and phenytoin are reversed compared to a conventional C18 reversed-phase separation (20). This is not surprising, however, considering the nature of the ISRP polyphenylalanine phase. Relative to phenytoin’s elution, the ISRP tripeptide phase appears comparable in retentive strength to a conventional phenyl phase and weaker in partitioning strength compared to a C8 and C18 column (Figure 6). This means that unnecessarily high concentrations of strong organic modifiers, such as methanol, in the mobile phase are not required to elute the analytes. It is important for the elution of proteins to be carried out under weaker solvent conditions, since methanol and other strong proton acceptor solvents, in high concentrations, tend to precipitate proteins. No precipitation of proteins is observed with the 20% acetonitrile mobile phase, giving an ample solvent strength range. Although much research remains to be completed, in order to produce an optimum ISRP HPLC column, the successful separation of these analytes on the first high-performance ISRP column demonstrates that the internal surface reversed-phase concept is suitable to facilitate the HPLC analysis drugs by direct injection of serum samples. Prototype 5-pm ISRP HPLC tripeptide columns made from Hypersil silica have withstood from 50 to 100 unfiltered serum sample

Flgure 8. Chromatographic retentlon of phenytoln on the high-performance ISRP trlpeptlde column compared to a conventlonal phenyl column, C8 column, and C18 column of equal length: mobile phase, 20% acetonitrlle/33% methanol/47% 0.1 M phosphate (pH 6); flow rate, 1.0 mL/min; detection, 254 nm; sample size, 20 pL.

injections before losing efficiency. This implies that residual proteinaceous material was accumulating. Attempts to regenerate the columns by the removal of proteinaceous contaminants with strong mobile phases proved unsuccessful. The approximate 6% loss of protein on the ISRP Hypersil supports has been attributed to the large initial pore diameter of the Hypersil silica (123 f 30 A). This subsequently obtained information suggests that a significant percentage of the pores was larger than desired, even after the tripeptide derivatization. Preliminary results acquired from ISRP tripeptide columns produced from 5-pm silica with initial pore diameters of 80 f 30 8, support this hypothesis. These latter ISRP columns have enabled the injection of as many as 240 serum samples before performance loss occurred. Column regeneration and performance recovery have been achieved by the removal of the residual proteinaceous contaminants with stronger mobile phases. With the return to nondenaturing mobile phase conditions, injection of serum samples can then be continued for some, as yet undetermined, upper limit. The internal surface reversed-phase chromatographic concept, therefore, demonstrates that low-performance as well as high-performance silica supports can be designed to be exclusionary and appreciably nonadsorptive to proteins, while concomitantly enabling the hydrophobic partitioning of small analytes. Registry No. Gly-L-Phe, 3321-03-7; Gly-L-Phe-L-Phe, 13116-21-7;phenytoin, 57-41-0; phenobarbital, 50-06-6; carbamazepine, 298-46-4.

LITERATURE CITED (1) Hearn, M. T. W. Adv. Chr0matogr.~?982,20, 4-82. (2) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography”, 2nd ed.; Wiley-lnterscience: New York, 1979; Chapter 17. (3) Analytichem International, Current 1983, 2, 5-12. (4) Roth, W.; Beschke, K.; Jauch, R.; Zimmer, A,; Koss, F. W. J . Chromatogr. 1981, 222, 13-22. (5) Nazareth, A.; Jaramillo, L.; Karger, B. L.; Giese, R. W.; Snyder, L. R. J . Chromatogr. 1984, 309, 357-368.

Anal. Chem. 1985, 57, 1763-1765 (6) Juergens, U. J . Chromatogr. 1984, 370, 97-106. (7) De Jong, G. J. J . Chromafogr. 1980, 183, 203-211. (8) Hux, R. A.; Mohammed, H. Y.; Cantwell, F. F. Anal. Chem. 1982, 54, 113-117. (9) Andereg, J. W. J . Am. Chem. SOC. 1955, 77, 2927. (10) Yau, W. W.; Kirkland. J. J.; Bly, D. D. "Modern Size-Exclusion Liquid Chromatography"; Why-Interscience: New York, 1979; Chapter 2. (11) Unger, K. K.; Kinkel, J. N.; Anspach, 6.; Gleshe, H. J . Chromatogr. 4QfIA .- - ., -298 - - , 3-14. - . .. (12) Schmldt, D. E.; Giese, R. W.; Conron, D.; Karger, 8 . L. Anal. Chem. lQB0. 52. 177-182. ----, (13) Pinkerton, T. C.; Hagestam, I . H. US. Patent, 6646 153, 1984. (14) Bethell, G. S.; Ayerer, J. S.; Hancock, W. S.; Hearn, M. T. J . 8/01. Chem. 1979, 254, 2572-2574. Glad, M.; Hasson, L.; Mansson, M. 0.;Ohlson, S.; Mos(15) Lasson, P. 0.; bach, U. Adv. Chromatogr. 1983, 27, 41-85. (16) Hofman, K.; Bergman, M. J . 8/0/.Chem. 1940, 734, 225. (17) Slggia, S. "Quantitatlve Organlc Analysis vla Functional Groups"; Wiley-Interscience: New York, 1949; pp 8-9.

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(18) Smith, G. F. "Analytical Appllcations of Periodic Acid and Iodic Acid"; G. F. Smith Chemical Company: Columbus, OH, 1950. (19) Stout, S. A.; Devane, C. L. J . Chromatogr. 1984, 285, 500-508. (20) Gerson, 6.; Bell, F.; Chan, S. Clin. Chem. (Winsfon-Salem, N . C . ) 1984, 30, 105-108. (21) Haroon, V.; Keith, D. A. J . Chromatogr. 1983, 276, 445-450. (22) Lunde, P. K. M.; Rane, A.; Yaffe, S. J.; et al. C/h. Pharm. Ther. 1970, 1 1 846-855. (23) Miller, T. D.; Plnkerton, T. C. Anal. Chim. Acta, In press. I

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RECENEDfor review January 28,1985. Accepted April 9,1985. The authors wish to express their appreciation to the Foremost-McKesson Foundation, Inc., for funding this research, through the Cottrell Research Grant Program of the Research Corporation.

CORRESPONDENCE Diffusion Layer Imaging: Spatial Resolution of Electrochemical Concentration Profiles Sir: We report here a spectrophotometric technique for determining concentration vs. distance profiles of absorbing solution species generated at an electrode surface. The technique can accurately describe diffusion layers as thin as 8 pm, after only 50 ms of electrolysis. In this paper, we describe the apparatus and its performance and discuss its potential for providing new information about mass transport and reaction mechanisms. The fundamental importance of mass transfer to electrochemistry has spawned a variety of theoretical and experimental examinations into diffusion (1,2), convection (3-51, and migration (6) as mechanisms for the transport of redox species t o an electrode. For several well-defined mass transport situations, the Faradaic current may be predicted from theories of diffusion and hydrodynamics, but in many cases solutions are not readily available. Diffusion to microelectrode arrays (7,8),mass transport in flowing streams (9, IO), and mixed convection/migration conditions are examples of cases where concentration vs. distance profiles are not available, and the Faradaic current is not accurately predictable from theory. Several approaches have been pursued to experimentally observe concentration- vs. distance profiles near an electrode, including interferometric methods (10-15) based on refractive index gradients near an electrode and on UV-vis absorption by electrogenerated species (16-18). The present approach is a spatidy resolved UV-vis absorption measurement which permits concentration vs. distance profiles to be obtained with better resolution and shorter time scales than those from previous methods. The cross section of a beam passing parallel to a planar electrode surface is magnified and imaged onto a photodiode array detector. Each diode samples a discrete distance from the electrode, and Beer's law may be used to directly determine a spatially resolved concentration profile. EXPERIMENTAL SECTION The cell, electrodes, and chemical systems were identical with those described previously for diffractivespectroelectrochemistry (19). Trianisylamine (TAA) in acetonitrile was oxidized at a platinum electrode to TAA+. at +0.8 V vs. aqueous SCE. The 0003-2700/85/0357-1763$01.50/0

diffusion coefficient of TAA (1.25 X 10" cm2/s) and molar absorptivity for TAA+. (11OOO & 200 M-' cm-', at 633 nm) have been reported previously (19). The optical apparatus shown in Figure 1 consists of a 2Ox magnifier producing an image of the beam cross section on the face of a 1024 element photodiode array. L1 (f = 1.48 cm), L2 (f = 5.0 cm), and a 25-pm pinhole form a collimated, spatially filtered, 2.9 mm diameter beam which passes parallel to a planar electrode surface made by polishing the edge of a Pt sheet (19). L3 (Mellis Griot LAO 126,f = 10 cm, diameter = 3.15 cm) and L4 (Space Optics Research Labs Fourier lens, f = 38 cm, diameter = 7.6 cm) form a magnifier with f = 8.67 cm. A photodiode array detector (Tracor 6112) was positioned vertically at the image plane of the magnifier, such that each element monitored a segment of the magnified beam cross section. The magnification was calibrated by using gold minigrids of known dimensions (Buckbee-Mears)and in the work reported here had a value of 20.0. The system was aligned and focused with the array scanning, with the criterion for optimum alignment being the sharpness of the magnified electrode edge. For 20X magnification, four 25 pm wide diodes fell on the rising portion of the electrode edge, indicating an apparent transition from electrode to solution of about 5 pm. On the basis of existing theories for the imaging of an edge using coherent light (20), the true electrode edge at the image plane was taken to be 25% up the rising portion of the image. This point was used as the origin of the distance axis in all plots. The Tracor Northern diode array system and PAR 173 potentiostat were triggered by an Apple 11+ microcomputer, which also stored digital data from the Tracor. The accuracy of the photodiode array was checked by using a range of calibrated neutral density filters, and absorbance error was less than f1.5% over the the absorbance range employed. For the electrochemical experiments, the array was scanned continuously, and the scans immediately before electrolysis and at the desired time after electrolysis began were stored and used to determine the absorbance as a function of channel number. The distance from the electrode sampled by a given photodiode, indicated here as x,, was calculated from the channel number by multiplying by the factor 1.25 pm/channel(25 pm divided by 20X magnification). The concentration of T U + .at a particular distance was calculated from the absorbance at that distance using Beer's law, with the path length equal to the electrode length along the optical axis (0.0150 cm). In all references to the diffusion layer thickness, it is defined as the value of x , where the T U + . concentration equals 50% of the bulk TAA concentration. 0 1985 American Chemical Society