Automated, High-Resolution Analyses for the Clinical Laboratory by Liquid Column Chromatography

AUTOMATED. HIGH-RESOLUTION ANALYSES FOR THE CLINICAL LABORATORY BY LIQUID COLUMN CHROMATOGRAPHY

.

Charles D Scott Biochemical Technology Division. Oak Ridge National Laboratory' Oak Ridge. Tennessee

1. Introduction ......................................................... 2. Analyticalsystems .................................................... 3. Description of Analyzers............................................... 3.1. General System Description...................................... 3.2. Separation Systems............................................. 3.3. Eluent Delivery ................................................ 3.4. Generation of the Eluent Concentration Gradient ................... 3.5. Sample Introduction ............................................ 3.6. Column Monitor ............................................... 3.7. Data Reduction ................................................ 3.8. UV-Analyzer ................................................... 3.9. Carbohydrate Analyzer .......................................... 3.10. Ninhydrin-Positive Compound Analyzer........................... 3.11. Organic Acid Analyzer .......................................... 4. Experimental Results and Applications., ................................ 4.1. Chromatographic Results ........................................ 4.2. Identification of Separated Constituents ........................... 4.3. Normal Values................................................. 4.4. Differencesin Pathological States and During Drug Intake .......... 5. Utility and Future of High-Resolution Analytical Systems ................. 5.1. Data Processing................................................ 5.2. Clinical Significance............................................. 5.3. Economics of High-Resolution Analyses ........................... 5.4. Screening Laboratories.......................................... 5.5. Other Uses ..................................................... References...............................................................

1 3 4 4 4 8 9 10 10 11 11 16 18 22 25 25 27 32 35 36 37 37 37 39 39 39

1 . Introduction

Many analytical methods used in the clinical laboratory today result in the analysis of a single constituent or of a single group of constituents in a physiological sample mixture . In most of these analytical procedures. an attempt is made to quantify the constituent without isolating it from the complex mixture . A great deal of developmental effort has been directed toward mechanizing many of these methods and, in some cases. in combining several analyses into a single. complex. automated instrumental array that requires a minimum of operator time . Although this 'Operated for the U . S. Atomic Energy Commission by Union Carbide Corporation. 1

2

CHARLES D. SCOTT

developmental work has been extremely important to the clinical laboratory from the standpoint of economics, recent research in the medical sciences will probably lead to even more drastic changes in the clinical laboratory in the near future. It is now apparent that many pathological states will ultimately be defined, studied, and treated on the molecular level. There is a considerable body of information that suggests that the levels of chemical constituents in various body fluids can be used to help indicate bodily function and malfunction. This is not a new concept for the clinical laboratory, but the number of these potential “chemical indicators’’ has been expanded to several hundred. For example, in a recent bibliography ( K l ) on urinary constituents, the literature for a three-year period has over 3000 citations to over 700 molecular constituents, many of which could have pathological significance. Quantitative methods for analyzing for large numbers of the individual constituents of body fluids have frequently involved several steps and excessive operator time. As a result, such complex analyses have been relegated to the research laboratory. It would be extremely difficult and expensive for the clinical laboratory to use these methods on a routine basis, even if they could be entirely automated. However, new highresolution analytical systems that are capable of automatically analyzing for many of the individual constituents of a physiological sample may be useful in the clinical laboratory for such an in-depth analysis. The term “high-resolution analysis” has been chosen to describe an analysis in which a large number of all the constituents of a sample mixture are separated and quantified. Thus, high-resolution analytical techniques have two very necessary components: (1) a means of separating the individual components; and (2) a means of detecting and quantifying the separated components. In general, the separation techniques that have proved most satisfactory have been some form of chromatography or electrophoresis, and quantification has been achieved primarily by photometric monitoring for liquid systems and flame ionization for gaseous systems. Relatively few truly automated, high-resolution analytical systems are now used in the clinical laboratory. For this presentation, I have arbitrarily chosen only those systems that use column chromatography for separation. This choice is based not only on the ability of these systems to separate literally hundreds of the molecular constituents in a physiological fluid but also because they are directly amenable to a high degree of automation. Obviously, this latter point is extremely important for any future development in the clinical laboratory. Further, only liquid chromatography will be discussed here since there has recently

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

3

been an excellent review of the use of gas chromatography in the clinical laboratory (S10). It is difficult to establish the time, places, and pertinent investigators involved in developing high-resolution analytical systems based on liquid chromatography since this technology has been evolving for many years. Yesterday’s high-resolution systems are now considered very lowresolution systems indeed. Certainly the early work of Cohn in separation of nucleic acid derivatives by ion-exchange chromatography (C2) was important, as was the development of an automated analytical system for amino acids by Moore and Stein ( M I ) . Hamilton showed that literally hundreds of ninhydrin-positive compounds in urine could be separated and quantified by a modified amino acid analyzer (H3), and Anderson and others followed through on some of Cohn’s work to automate the analyses of complex biological fluids in a single system (Al, Sl). There are a t present many investigators involved in the general area of high-resolution analysis for the clinical laboratory. Many recent contributions in this field can be found in the proceedings of the annual symposium series on “High-Resolution Analyses and Advanced Analytical Concepts for the Clinical Laboratory” (S4, S6, 58). 2.

Analytical Systems

Although the concentrations of the constituents of all types of body fluids represent potentially useful diagnostic information, analysis of the most complex body fluid, urine, presents the most ambitious challenge. One of the most severe tests for the utility of a high-resolution system is its usefulness in analyzing for the constituents of urine. This body fluid has long been neglected in the clinical laboratory. The four analytical systems that will be considered here are at least potentially useful for urine analysis as well as for the other less complex body fluids. They are primarily used for the analysis of the low-molecular-weight (less than 1000) constituents. Two of these systems, an analyzer for the UV-absorbing constituents (UV-analyzer) and one for carbohydrates, will be discussed in some detail. Two others, one for ninhydrin-positive compounds (amino acids and related compounds) and an analyzer for organic acids, will be introduced as systems that have great potential but which have not been fully developed as yet. These four analytical systems certainly do not represent all the concepts for the use of liquid chromatography in body fluids analysis; however, they are systems that have been used a t least to some degree in clinical and medical research laboratories. The UV- and carbohydrate analyzers were specifically developed to

4

CHARLES D. SCOTT

be used for analyzing body fluids, and prototype systems of each analyzer are now being used a t several laboratories. On the other hand, the ninhydrin-positive and organic acid analyzers were not originally developed to be used for complex body fluids, but rather for much simpler mixtures, e.g., protein hydrolyzates. As a result, these two systems have not been fully exploited for body fluids analyses, particularly for urine analysis, although preliminary work indicates that they may have great utility. Thus, the latter two systems will not be discussed in as much detail as the UV- and carbohydrate analyzers. 3.

Description of Analyzers

Up to this point in time, high-resolution liquid chromatography requires the use of very small sorption particles packed in relatively long columns. This results in the necessity of operating with relatively high column inlet pressures to force the eluent through the column a t a reasonable rate. This requirement of high-pressure operation is the major difference between high-resolution systems and the more conventional liquid chromatography. Much of the following discussion will emphasize the high pressure requirements. 3.1. GENERALSYSTEM DESCRIPTION

Automated liquid chromatographs contain the following major components: (a) the separation section, which consists of a closed tubular column packed with small particles of the solid sorbent or support material; (b) an eluent storage and, in some cases, an eluent gradient preparation section; ( c ) an eluent delivery system equipped t o deliver the eluent to and force i t through the separation column; (d) a means for introducing the sample to the column; and (e) a means for detecting and quantifying the separated constituents in the column eluate (see Fig. 1). Automated data acquisition and processing may also be used. The requirements of high-pressure operation affect the design and operation of the eluent delivery, sample introduction, and separation systems. Many of those involved in developing high-resolution analytical systems for body fluids have made very significant contributions to high-pressure liquid chromatography technology.

3.2. SEPARATION SYSTEMS The most important component of the liquid chromatograph is the separation system. Recent advances in liquid chromatography have included the development of many new types of sorption media that have made high-resolution separations possible.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

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FIQ.1. Liquid column chromatography.

3.2.1. Separation Media

The aim in recent developments has been to produce media in which the solid-phase mass transport resistances are reduced. A reduction in these resistances will allow the chromatographic system to operate closer to equilibrium conditions, and should result in faster and more effective separations. All the systems under consideration here achieve high resolution by using relatively small particles (down to about l o p diameter) in the stationary sorption phase in chromatographic columns up to about 150 cm long. The small particles are used to reduce the solidphase diffusional effects, and the relatively long columns are necessary to provide a sufficient number of separation stages to achieve the high resolution. 3.2.2. Pressure Drop

The combination of small particles and long columns contributes to high operating pressures. The effects of column and operating parameters on the pressure drop of liquid-chromatography columns designed to operate a t pressures less than about 100 psi can essentially be disregarded since design problems are minimal ; however, these effects become very important in high-pressure chromatography (greater than lo00 psi). For a particular type of sorption medium, the major parameters that influence the pressure drop across an ion exchange column are: particle diameter, flow rate, column length, and fluid properties such as density and viscosity. These effects have not been thoroughly studied for small particles; however, previous data (H2) and some of the author’s recent work have shown that the pressure drop across a packed column is inversely dependent on the square of the mean diameter of

6

CHARLES D. SCOTT

FIQ.2. Pressure drop across ion exchange resin columns as a function of flow rate for R S ~ Mof different particle size. Operating conditions: 40°C; column, 0.62 X 100 cm, stainless steel; resin, Dowex 1 X -8.

ion exchange resin particles and linearly dependent on the linear velocity of the liquid phase and the length of the column (Fig. 2).

3.2.3. Columns Metal columns, which can be easily fabricated from seamless metal tubing, can be used for high-pressure techniques. Conventional compression tubing fittings can be used for the fluid entrance and exit and for holding a porous metal support for the fixed bed (Fig. 3). Although the use of precision-bore tubing may be slightly more advantageous, good results have been obtained with common seamless tubing. Some glass columns operable to about 1000 psi are available and have been used in early models of the systems under consideration. 3.2.4. Column Geometry The geometry of a chromatographic column has a significant effect on the resolution that is achieved. As the length of a column is increased, the separation of two components becomes more efficient ; however, the width of the peaks is also increased. The diameter of the column should not have a great effect on resolution (assuming that comparable flow velocities and a proportionally scaled sample size are used) as long as

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

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FIO.3. High-prearmre chromatographic coIumn fabricated from stainless steeI tubing. From Scott (512) copyright @ 1968 Clinical Chemistry.

the column is sufficiently small to prevent radial variations in fluid properties but not small enough to require a sample of such limited volume that the separated solutes cannot be detected by the column monitoring system. Column diameters in the range of 0.15 to 0.60 cm

8

CHARLES D. SCOTT

have been found suitable for analytical purposes. Column lengths up to 200 cm have been used effectively. 3.3. ELUENT DELIVERY Two basic types of eluent delivery systems are used in liquid column chromatography. These are constant-flow devices and pulsating pumps (Fig. 4). Examples of the former include constant-drive syringes and reservoirs with gas overpressure, and the latter include reciprocating piston pumps. All the systems described here have been designed to use piston pumps with pulsating flow, although it would be possible to design such systems with constant-flow devices. It should be pointed out that in systems with a column pressure drop in excess of 1000 psi, pulsating pumps are sufficiently accurate metering devices with flow variations of less than 10% during each pulse cycle. I n general, pulsating pumps are less expensive and somewhat more simple to use in chromatographic gystems. They are particularly advantageous when gradient elution (i.e., an eluent composition that changes CONCENTRATED BUFFER

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ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

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with time or elution volume) is used, since the gradient can be developed prior to contact with the high-pressure environment (Fig. 4). At pressures above about 3000 psi, i t is difficult to maintain a good mechanical seal around a moving piston. The difficulties are usually more pronounced for the pulsating pump since its plunger moves more rapidly and more frequently than the constant-flow devices. This disadvantage has now been partially circumvented by the development of the diaphragm-plunger pulsating pumps in which a pulsating plunger delivers a hydraulic fluid to a sealed diaphragm in contact with the eluent. The eluent is pumped by the movement of the diaphragm, and this arrangement abolishes the need for a high-pressure seal. Such pumps are used successfully in the W- and carbohydrate analyzers. 3.4. GENERATION OF

THE

ELUENT CONCENTRATION GRADIENT

Gradient elution chromatography is a very powerful and frequently necessary technique when complex mixtures are being separated. Increasing the concentration of a buffer with time or elution volume decreases the distribution coefficients of the more strongly sorbed species, thus allowing the elution time to be significantly decreased without jeopardizing the separation of the less strongly sorbed species a t the beginning. Changing the pH or some other eluent property also allows a more efficient separation. Nearly any type of continuous eluent gradient can be generated by connecting two or more chambers containing solutions of different properties to a common mixing chamber (Fig. 4 ) . (See also the description of UV- and carbohydrate analyzers.) The eluent properties of the fluid stream from such a system vary with the volume removed, depending only on the relative cross-sectional areas of the chambers and the properties of the fluid being used as the eluent. Typically, operation is initiated by filling each chamber until overflow occurs. Then, as the run progresses, the eluent properties change due to the changing cross-sectional areas of the chambers. At the end of the run, a reservoir connected to the bottom of the chamber containing the initial eluent automatically equilibrates the column with the starting eluent in preparation for the next run. A stepwise eluent gradient can be generated by simply using a series of reservoirs with different eluent solutions all connected to the pump feed line and each line being actuated by a solenoid valve. (See description of the ninhydrin-positive compound analyzer.) This technique works well if the step changes do not upset the monitoring device; however, it necessitates additional equipment.

10

CHARLES D. SCOTT

3.5. SAMPLE INTRODUCTION The most effective method for introducing a sample into an automated chromatographic system is to feed it directly into the eluent line just before the latter contacts the chromatographic column. A hypodermic syringe entering a septum connected to the eluent line may be used to accomplish this; however, in high pressure operation this will usually necessitate stopping the eluent flow so that the septum and syringe are exposed to a reduced pressure. The UV- and carbohydrate analyzers use a sample injection valve that contains six ports, each pair of which is interconnected. I n one orientation of the valve, a sample can be loaded into the sample loop, which becomes a part of the eluent line when the ports are reoriented (by turning the valve handle) (Fig. 5 ) . Valves that allow automated sample introduction a t pressures up to 5000 psi without interrupting the eluent flow have been developed and are now available commercially (S2). 3.6. COLUMN MONITOR

In all four systems, the eluate stream transports the separated constituents of the sample mixture to flow monitors that are either a photometer (for the UV-analyzer) or a colorimetric detector (for the other systems). In the latter case, reagents are mixed continuously with the

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FIO.5. Use of a six-port valve to inject a sample into the eluent stream of a chromatograph. From Scott (S11) with permission.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

11

eluate stream and the resulting reaction mixture is continuously monitored by a flow colorirneter. When colorimetric monitoring is used, additional process variables have to be considered. These result from the necessity of introducing a metered stream or streams of reagent into the eluate stream, mixing the two streams thoroughly, allowing the necessary chemical reaction to occur between the separated constituent and the reagent, and continuously monitoring this reaction stream with a colorimeter. For systems in which large reagent flow rates (greater than 10 ml/hr) are used, this can be done by metering the reagent streams with positive displacement pumps. When pulsating pumps are used, the variation in flow rates must be reduced by suitable damping devices. For systems that require very low reagent flow rates, and even for larger flow rates, a successful reagent metering system can be designed to include a reagent reservoir with near-constant overpressure or hydrostatic head coupled with a controlled flow resistance, for example, narrow bore tubing or a control valve ( J l ) . Rotameters can be used t o monitor the actual flow rate. If the reagent hydrostatic head or gas overpressure remains essentially constant during the course of a run, the reagent flow rate will remain relatively constant even a t a flow rate of a few milliliters per hour. 3.7. DATAREDUCTION All the systems discussed here use conventional strip chart recorders for recording the photometer or colorirneter output, and the resulting record is a conventional histogram in which the absorbance of the eluate or eluate-reagent reaction mixture is recorded as a function of time. I n addition, some prototype systems of the UV- and carbohydrate analyzers use on-line computers for data storage and processing ( C l , 57). I n any case, the area of each chromatographic peak is directly related to the quantity of material represented by that peak. Quantification of the chromatographic data is achieved either by graphical (strip chart recorder) or numerical (on-line computer) integration of each chromatographic peak to obtain the peak area. Where there are mutually interfering chromatographic peaks, the resulting absorbance envelope must be convoluted into its individual peaks. This is most easily done by the on-line computer using conventional spectral stripping techniques ( C l , 57). 3.8. UV-ANALYZER The present model of the UV-analyzer will provide the basis for analytical systems that can be used routinely in the future (Pl, 55).

12

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FIG.6. Automated, high-resolution chromatograph for analyzing for the Wabsorbing constituents in body fluids.

Several prototypes of this analyzer are currently being tested at various clinical and medical research laboratories.2 The analyzer uses a heated, high-pressure (up to 4000 psi) anion exchange column, concentration gradient elution with an aqueous acetate buffer for separation and transport of the constituents of the sample mixture, and a recording photometer for detection and quantification of the separated constituents (Fig. 6 ) . Earlier models of this analyzer were housed in standard 24 X 24 X 63 in. cabinets (Fig. 7); however, miniaturized versions with capillary separation columns are now being used (Fig. 8 ) . An anion exchange resin produced by Bio-Rad Laboratories (Aminex A-27) in the size range of 10-15p has been found to be satisfactory. 'Construction prints of the earlier models are available as CAPE-1753 from the National Technical Information Service, U. S. Department of Commerce, 5285 Port Royal Road, Springfield, Virginia 22151.

ANALYSIS BY LIQUIDCOLUMN CHROMATOGRAPHY

13

FIG.7. W-analyzer prototype Mark 11. From Scott (55) with permission.

The separation columns are fabricated from standard type 316 stainless steel tubing that is either 0.22 or 0.62 cm ID (depending upon whether it is a n advanced miniaturized system or an earlier model) and 150 cm long. A 1 in. OD stainless steel heating jacket surrounds the column. The ion exchange resin is packed into the column as a thick slurry using a dynamic loading technique which provides reproducible

14

CHARLES D. SCOTT

Fra. 8. Miniaturired Mark 111-A UV-analyzer. From Pitt (Pl), copyright @ 1070 Clinical Chemistrg.

loading from column to column (53).An ammonium acetate-acetic acid buffer (pH 4.4) whose concentration varies from 0.015 to 6.0M during the course of the analysis is used as the eluent, and the separation column is maintained a t 25°C for the first 30% of the run and a t 60°C thereafter by a heated circulating fluid.

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The detector is a miniature, recording, dual-beam UV flow photometer operating continuously a t two different wavelengths, 254 and 280 nm (Tl,T2). The dual-beam mode of operation provides a means of referencing the changing properties of the eluent stream by differentially comparing the eluent stream to the eluate stream. Samples are introduced by a six-port injection valve, and analytical results are presented graphically as a chromatogram showing the UV absorbance of the eluate stream versus run time, each molecular constituent being represented by a chromatographic peak (Fig. 9). The required sample size is 0.1-0.5 ml, and the total separation time is 40 hours for the larger system and 24 hours for the miniaturized system. Sensitivity is a few nanograms for many constituents (Fig. 10).

3.9. CARBOHYDRATE ANALYZER The carbohydrate analyzer also uses a heated, high-pressure anion exchange column of the same design and utilizing the same resin as that used for the UV-analyzer; concentration gradient elution with a borate aqueous buffer; and detection and quantification by a continuous colorimetric system (Figs. 11 and 12) (K2,S6).s Miniaturized versions using capillary columns are also now being used. The borate buffer is necessary to complex the neutral carbohydrates to give them ionic properties that then allow separation by anion exchange chromatography. A sodium tetraborate-boric acid buffer (pH 8.5) whose composition varies from 0.169 to 0.845 M in the borate ion is used as the eluent. The anion exchange separation column is maintained at a constant 55°C. Carbohydrate detection is by the continuous colorimetric reaction of sulfuric acid and phenol with the carbohydrates in the eluate. T o accomplish this, the system includes: (1) a reaction column into which the eluate and reagents (5% phenol solution and concentrated sulfuric acid) are continuously metered and mixed; (2) a reaction section maintained a t 100°C through which the reaction mixture flows; and (3) a flow colorimeter that continuously measures the absorbance of the reaction mixture a t wavelengths of 480 and 490 nm (Fig. 11). The reagents are metered into the reaction column by using controlled pressure or hydrostatic head in the reagent reservoirs, a fixed pressure drop across a length of capillary tubing, and a control valve in the reagent lines ( J l ) . Rotameters are used to measure the reagent flow rates. 'Construction priuta of the earlier models are available as CAPE-17'19 from the National Technical Information Service, U. 8. Department of Commerce, 5285

Port Royal Road, Springfield, Virginia. !22151.

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FIQ.11. Automated, high-resolution chromatograph for analyzing for carbohydrates in body fluids.

Samples of 0.5 to 12 ml are introduced by a six-port injection valve, and the resulting chromatogram is a measure of the absorbance of the eluate reaction mixture as a function of time (Fig. 13). Separation time is 20 hours. 3.10. NINHYDRIN -POSITIVE COMPOUND ANALYZER

The modern amino acid analyzer is one of the most highly developed liquid chromatographs now being routinely used in the research laboratories. It is also used to some extent for analysis of physiological fluids, mainly serum ( E l ) . However, the resolution of such systems does not approach that which has been previously demonstrated, especially for urine analysis. Such a high-resolution analyzer has a great potential for the clinical laboratory.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

19

FIG.12. The Mark I1 carbohydrate analyzer. From Scott (55) with permission.

Many different experimental systems for analysis of amino acids have been described, but the most successful from the standpoint of highresolution analysis of physiological fluids is the system described by Hamilton in which he was able to separate a t least 175 components in human urine (H3) using a single cation exchange column system. This

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FIG.13. Typical chromatograms from the carbohydrate analyzer showing the difference between urine and blood serum and the identification of some of the chromatographic peaks. Sample sizes: sugar reference compounds (top), 0.62 p M except 125 p M melibiose and 490 nm, --- 480 nm.) From Scott (S5) with glucose-l-POa; urine (middle), 12.4 ml; and blood serum (bottom), 1.6 ml (permission.

22

CHARLES D. S C O W

FIQ. 14. High-resolution cation exchange chromatography of ninhydrin-positive compounds in body fluids. From Hamilton (Hl), with permission.

system is composed of a high-pressure glass column 0.636 X 135 cm containing the ion-exchange resin that is temperature controlled by circulating fluid ; a positive displacement piston pump for eluent delivery, with stepwise buffer change being controlled by a series of solenoid valves connected to the pump inlet manifold; and a ninhydrin colorimetric development system in which the ninhydrin-positive compounds in the column are reacted with a stream of a ninhydrin reagent followed by colorimetric monitoring a t 440 and 570 nm (Fig. 14) ( H l ) . The small-diameter ion exchange resin that was used (Aminex A-7, 10 2 2 p ) necessitated relatively high operating pressures; however, the use of a glass column necessitated a pressure limitation of I000 psi or less. This resulted in an operating time of as much as 65 hr for a single urine analysis. In Hamilton’s early work, the sample was placed on the ion-exchange resin by removal of liquid a t the top of the column and injection of the sample directly onto the top of the resin bed while the eluent flow was stopped. This is an adequate means of sample introduction, although an automated system can probably also be used. The chromatogram was developed with the stepwise elution by sodium citrate buffers of varying concentrations and pH from a typical sample of 0.5 ml of the body fluid (Fig. 15).

3.11. ORGANICACID ANALYZER An organic acid analyzer for physiological fluids has not been developed to the same degree as the other systems. However, this type of analysis is of sufficient importance that it has been included in this pres-

23

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

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FIa. 15. High-resolution chromatogram of the ninhydrin-positive compounds in 0.5 ml of human urine. This was a single cation-exchange separation using step elution that required 65.5 hours. From Hamilton (Hl, H3), Handbook of Chemktry,

2nd Ed., p. B-92, with permission.

entation. Several workers have attempted to use organic acid analyzers for determining organic acids in physiological fluids. Typical of these is the system used by Rosevear e t al. ( R l ) , which probably has the highest resolution and sensitivity reported for analysis of organic acids in physiologic fluids. Rosevear’s system is an extensively modified version of a commercial instrument (Fig. 16). It uses a temperature-controlled glass chromatographic column (175 cm X 0.4 cm) operating a t 20°C with eluent pressures up to 1000 psi; pulsating piston pumps for eluent delivery and colorimetric reagent metering; and a continuous colorimetric monitoring of the eluted organic acid by mixing and reacting an indicator reagent with the column eluate, followed by continuous detection with a flow colorimeter. The separation medium is activated silicic acid with a particle size of 10-40p, which is packed into the column by a dynamic introduction of a slurry. Unfortunately, a new column must be packed for each analysis. This is a weak point in the system, and it is an obvious area

24 CHARLES D. SCOTT

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ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

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for future development. It should be pointed out that a few years ago many liquid chromatographic separations were operated in a similar mode. The eluent stream is a mixture of chloroform and tert-amyl alcohol with the addition of a small amount of water for adjusting the activation of the silicic acid. A multichamber gradient generation system was used to vary the eluent organic solvent makeup from essentially pure chloroform to a 1:l mixture of the two solvents, The colorimetric monitoring system used an ethanol solution of the indicator, neutral red (3-amino-7-dimethylamino-2-methylphenazine), that is mixed continuously with the column eluate and then monitored by a flow colorimeter a t 550 nm. Although the organic acid fraction of physiological fluid samples can be introduced into the system in several ways, one means is to presorb the sample on silicic acid, and then add this sorbent to the top of the column after which the gradient elution is started. This necessitates an additional manual operation that also presents a future area for development. A typical analysis requires 0.1 to 0.2 ml of the body fluid sample with an analysis time of about 6 hours (Fig. 17). 4.

Experimental Results and Applications

High-resolution analyzers have been used to determine the molecular constituents of urine and blood serum as well as other body fluids, such as cerebrospinal fluid, perspiration, saliva, and amniotic fluid. Well over 300 molecular constituents can apparently be separated by a combination of all four types of analyzers; however, many of the separated components have not actually been isolated and identified by spectral and chemical tests. 4.1. CHROMATOGRAPHIC RESULTS

The UV-analyzer normally separates 100-120 chromatographic peaks from a urine sample in a 24-hour run (Fig. 9) (55); however, as many as 140 peaks have been separated from a single urine sample, and over 180 different components were separated from urine that had been concentrated by a sorption process (M3). Sensitivity levels of less than a microgram are observed for many components (Fig. 10). The carbohydrate analyzer has separated as many as 48 chromatographic peaks from a single body fluid sample ; however, chromatograms from urine samples of normal subjects have 30-40 peaks (Fig. 13) ( S 5 ) . The carbohydrate analyzer is sensitive to a few micrograms of each individual carbohydrate. The common amino acids are well separated by the conventional amino

26

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180 J

360

FIQ.17. Chromatogram of organic acids in 0.1 ml of human urine. From Rosevear (Rl), copyright @ 1970 Clinical ChemOtry.

acid analyzer, particularly those analyzers using two-column systems. When such systems are adjusted for physiologic fluid analysis, they separate 30-50 ninhydrin-positive peaks from serum (El) and about twice as many peaks from urine. In general, this requires an extensive increase in the analysis time. Although fewer analytical data are available on high-resolution analyses of the ninhydrin-positive components of body fluids using a single cation exchange column, a t least 175 such components have been isolated from urine, with the indication that perhaps additional resolution would result in additional peaks (Fig. 15). Submicrogram sensitivity has been demonstrated (H3).

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

27

There is an indication that the organic acid analyzer can provide meaningful resolution of more than 50 constituents in urine (Fig. 17) (R1). Again, the sensitivity will be less than a microgram for some components. In general, the components being separated and quantified by these analyzers are of relatively low molecular weight (less than a molecular weight of 1000). I n fact, the high-molecular-weight components are usually removed by ultrafiltration or precipitation for the ninhydrin-positive compound analyzer and, in some cases, for the other analyzers. The detected compounds are thus the metabolic and catabolic products of the life processes. Body fluids other than urine have considerably less complex lowmolecular-weight component spectrums, a t least a t the concentration levels that can be detected by these analyzers. For example, blood serum samples, when compared with urine, will have about one-fourth as many chromatographic peaks of UV-absorbing constituents and carbohydrates and about one-half as many ninhydrin-positive and organic acid chromatographic peaks. Cerebrospinal fluid appears to have about the same complexity in UV-absorbing and carbohydrate components as does blood serum, and perspiration falls somewhere between urine and serum. 4.2. IDENTIFICATION OF SEPARATED CONSTITUENTS

Actual identification of the separated body fluid constituents requires major experimental effort. Chromatographic peaks can be tentatively identified by comparing their chromatographic properties with those of reference compounds. However, confirmation of the identification requires isolation of the column eluate fraction represented by the chromatographic peak and determining the identity of the included constituent by chemical and spectral methods. The gas chromatograph and mass spectrometer have proved invaluable in this work. So far, the tentative chromatographic method has been used to make most of the identifications of the ninhydrin-positive and organic acid components, especially for urine constituents. This simply requires that the unknown peak has the same elution volume as a known reference compound. A significant effort has been made to provide more definite identifications for the components separated by the UV- and carbohydrate analyzers. To date, this has included over 70 UV-absorbing compounds and 18 carbohydrates, some of which are listed in Tables 1-3 (B2, M2). Tentative identification of many more compounds has been made in all four systems, and, hopefully, the efforts in confirmative identification will continue.

TABLE 1

SOME OF THE

COMPOUNDS SEPARATED FT~OM THE URINEOF NORMAL Swmxs BY THE W-ANALYZER BY GASCHROMATOGRAPHY AND Mass SPECTROMETRYO

W

Compound

Ureac Creatinine fl-Pseudouridinec UraciP 5.Acetylamino-s-a~~methylurscil" WMethyl-spyridone 5carboxsmide 7-Methylxanthinec 3,7-Dimethylxanihine Hypoxanthin@ Xantbine 3-Methylxanthine 1-Methylxanthinec Uric acidc

,x (-1

-a

232 262 261 263 258 269 273 249 267 269 267 276

Mu value for TMS

derivativej 12.44 15.57" 23.68 13.30 4

18.65 20.19 ---I

17.92 20.05 19.26 20.37 21.22

AND IDENTIFIED n

5

. l

Mass spectral datab

!i

Bsse ped

m/e (2)

m/e (3

m/e (4)

Mol. wt.

44

60 43 141 42 198 136 68 67 81 109 68 109 69

17 113 125 68 71 108 123 109 109 81 95 81 168

43 112 165 69 155 135 67 82 108 54 123 137 97

60 113 244 112 198 152 166 180 136 152 166 166 168

42 208 112 156 152 166 180 136 152 166 166 125

U

In

2-Amino-3-hydrox ybenzo ylgl y cine Phenylacetylglu tamine 4-Acetylaminobenzoylglycineh Etippuric acidc Citric acidc

3-Methoxy4hydroxybenzoylglycine 3-Methoxy-4-glucuronosidobenzoicacidh 3-Methoxy-4hydroxyphenylacetic acid. 4-Acetylsminobenzoic acidh PHydroxyhippuric acid 3-E thoxy4hydroxybenzoylglycineh 3-Hydroxyhippuric acid 3-Ethoq4glucuronosidobenzoic acid* 3-Methoxy-4-hydroxybenzoicacidcqh

258

4

258

23. 5OE

4

18.05 18.50 23.37

267 224

254 263 279 266 253 253 290 264 256

From Mrochek et al. (M2). Includes base peak and three next most significant m/e. 0 Reference compound available; data identical. Non-W absorbing. 6 MU value is for larger of two GC peaks. f Multiple-GC peaks indicate decomposition.

2

2

17.61 18.39 22.10

1

21.31 I

17.56

136 91 120 105 -* 151 168 137 137 121 137 121 154 168

121 187 162 77 225 151 182 120 195 165 93 137 153

2240 142

135

210 27f9 208 134

123 153 122 179 150 107 151 182 97

2399 358s 92 108 93 239 150 165 125

25ov

-

-

210 264 236 179 192 225 344 182 179 195 239 195 358 168

Methyl ester. Subject on artificial diet. Insufficiently volatile for MS; identified as TMS derivative with an integrated gas chromatograph-mass spectrometer. i Methylene unit values from 6 ft X 0.25 in. OD glass column packed with 3% GGSE-30 on 100/200 mesh Gas Chrom Q programmed from 100' to 325°C at 10°C/min. 0

s

E4

i3

z

E: D d

ii

z 0

3 x

c)

g

kE

W 0

SOMEOF

THE

COMPOUNDS SEPARATED FROM AND D IENTF IE ID

TABLE 2 URINEOF SUBJECTS WITH VARIOUSPATHOLOGIES BY THE GASCHROMATOGRAPHY AND MASS SPECFROMETBY"

THE

BY

W-ANALYZER 0

m

Compound n

Trigonellined.0 NicotinamideN-oxided.e Nicotinamided 1,7-Dimethylxanthine Allopurinold oxipurinol" 3-Methoxy-4hydroxyacetanilide 4Hydroxyacetanilide OrotidineJ 3-Methoxy4glucuronosidoacetanilide 4Glucuronosidoace~ilide Sulfanilamided Orotic acidd

16.12 18.43 18.92 17.92

1380 122 122 180 136 109 139 109

94 106 106 68 73 152 181 151

'139 78 78 123 135 52 124 81

95 138 94 95 109 53 96 95

242 240

2.i

A,<

2.i

A . i

-

-

-

263 276

21.53 17.50

172 68

156 156

92 113

108 69

264 268 262 263 249 253 244 242 266

2

17.21 14.55 2

2

A

137 138 122 180 136 152 181 151

288

357 327 172 156

3-Methoxy-4-hydroxymandelic acid 3-Methoxy-4-hydroxyphenyllactic acid Phenyllactic acidd CHydroxyphenylaceticacidd Benzoic acidd ZHydroxybenzoic acidd 2-Hydroxyhippuric acidd 4-Hydroxybenzoic acid 2,5-DihydroxyphenyIaceticacidd 2-Hydroxyphenylacetic acidd

279 272 259 278 272

302

300 250 295 274

18.85 20.25 15.85 16.28 12.30 15.14 20.88 16.28 18.40 15.75

153 124 91 107 122 120 121 121 122 134

137 212 148 166, 105 92 120 138 94 106

93 109 103 152 77

198 137 166 77 51

62 93 150 107

92 65 182i 78

138

64

198 212 166 152 122 138 195 138 168 152

From Mrochek et al. (M2). b Methylene unit values from 6 f t X 0.25 in. OD glass column packed with 3% GCSE-30 on l00/200 mesh Gas Chrom Q programmed from 100" to 325°C at 10°C/min. c Includes base peak and three next most significant m/e. Reference compound available; data identical. e Compound not previously reported in human urine. Multiple-GC peaks indicate decomposition. CJ Mass spectral data identical to standard recovered from buffer used in anion-exchangeseparation. h Insufficiently volatile for mass spectrometry. i Hydrolyzed sample gave same data &s parent compound. f Methyl ester.

rc

Em rc

E

8

Q

0

2K

Z

Q

31

L-d

?i

%

0 4 Ed

$

2

32

CHARLES D. SCOTT

TABLE 3 SOMEOF TEE COMPOUNDS SEPARATED FROM URINEOF NORMAL SUBJECTS BY THE CARBOHYDRATE ANALYZER AND IDENTIFIED BY GAS CHROMATOGRAPHY" MU valud Carbohydrate

Source

Peak 1

Sucrose

Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary Reference Urinary

27.46 27.42 27,34 27.31 18.61 18.57 18.60 18.57 16.47 16.45 16.71 16.75 18.68

Lactose Allulose Fructose Arabinose Fucose Galactose Sorbose Xylose Glucose

-

18.41

-

17.38 17.44 18.89

-

Peak 2

Peak 3

28.60 28.58 18.83 18.80 18.71 18.71 16.78 16.75 17.06 17.07 19.11 19.08 19.09 19.16 17.94 18.00 19.35 19.31

19.43 19.41 17.10 17.07 17.46 17.48 19.53 19.55 20.00 20.00

20.31 20.25

From Mrochek et al. (M2). Methylene unit values from the single or multiple peaks that result from the GC separation on 180 cm X 6.3 mm OD glass column packed with 5% SE-30 on 100/200 mesh Chromosorb W(HP) programmed from 100" to 325°C at 10"C/min. a

4.3. NORMAL VALUES For high-resolution techniques to have general utility, it must be established that the body fluids of normal subjects have a definable normal spectrum of chemical constituents and that various pathologic states can be associated with abnormal values of one or more of the constituents. UV and carbohydrate chromatograms from urine (24-hour composites) and serum samples from clinically normal subjects are very similar. About three-fourths of the major peaks are common to all the normal subjects tested, and the concentrations (peak sizes) are within relatively narrow limits (e.g., see Fig. 18) (52). Variation during the diurnal cycle is measurable but not prohibitive (Fig. 19), and variations during long periods of time are much less for one person than the variation from person to person (52).

W

U 4

'1h

I

I\

m

490 nm

W

U

U z

K

m a

m

0 In

wa

m 4

w

U W

4

a m a In 0

z

U

m a (0 0

a m

OFi 1.5.

Y 0 z

1 K 5: m

m

. -

!A

.

4

i i i 6 s i .is io

ii iz i3 i4 15 ELUTION TIME (HOUR)

,

h

16

(7

ii i 3 2 o

ELUTION TIME (HOUR)

1 W

U

Ft3

4

m

wa K

m

"6 W

w 0 z

m

4

5: m

2

z m

i

i i i 4

s

6 i i

9 io ii iz 13 i4 i5 ELUTION TIME (HOUR)

i6

i7

i8

19

20

1

wa K

a

Oo

i i

3

4 5

6 i ELUTION a 9

10

ii iz

i3

TIME (HOUR)

li is is

i7

m

is is

20

FIG.18. Typical urine carbohydrate chromatograms of eight normal subjects on random diets. From Jolley et al. (J3) with

permission.

TIME (Hours)

FIG.19. W chromatograms of the urine of normal subjects showing the effect of the diurnal cycle and the comparison between normal subjects. Run conditions: column, 0.45 cm ID X 200 cm 316 stainless steel with 10-p diameter Bio-Rad AG1-XS; urine samples, 2 ml each; temperature, 25°C increasing to 60°C after 15 hours; pressure, 1500-2300 psig; elution, sodium acetate-acetic acid b d e r at a p H of 4.4, varying in concentration from 0.015M to 6 M at an average flow rate of 28 ml/hour. (-, 260 nm; ----, 280 nm.) From Scott (S12), copyright @ 1968 Clinical Chemistry.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

35

Normal values can be altered by dietary factors, especially when unusual diets are used (e.g., synthetic diets such as Vivonex) (Y2), and by the ingestion of drugs (K3). However, these effects can be predictable, and nominal control over food and drug intake is sufficient to allow establishment of base-line chromatograms. Normal subjects on identical diets produce chromatographic patterns that are almost superimposa ble. 4.4. DIFFERENCES IN PATHOLOGICAL STATES AND DURING DRUGINTAKE

Significant differences have been noted between normal and pathological urine chromatograms. For example, the urine chromatogram of a patient with a neuroblastoma had very large homovanillic acid and vanillic acid peaks, indicating that these excretion products may be useful indicators of that pathological state (53). The lack of hippuric acid was noted in the urine of a patient with the Lesch-Nyhan syndrome (Fig. 20) (53).This, coupled with an increase in benzoic acid excretion, indicated that the glycine conjugation mechanism may have been impaired in that pathological state. These two examples show the utility of the “spectral approach” or establishment of the “chemical profile” of the body fluids that can now be achieved with high-resolution analyzers. Chemical indicators of abnormal states can be found without prior knowledge of their existence and, thus, without having to decide which specific chemical indicators are to be investigated. 1.00.8

0.6 -

HI PPURIC ACID

I

700

800

1

900 ELUTION VOLUME (ml)

I

1000

PIC.20. Comparison of a selected portion of the U V chromatograms of a normal reference urine (- --; A, 260 nm) and urine from an individual with the LeschNyhan syndrome (-; A, 260 nm). From Jolley (J3), copyright @ 1970 Clinical Chemistry.

36

CHARLES D. SCOTT

Allopurinol, an analog of hypoxanthine, is widely used in the treatment of hyperuricemia and gout. The drug is a potent inhibitor of xathine oxidase, which is the enzyme catalyzing formation of uric acid, and thus it decreases endogenous synthesis of this purine. Researchers using the UV-analyzer found that the drug and its metabolite had a previously unknown side effect, namely, the inhibition of endogenous pyrimidine synthesis (K3). This resulted in a large increase in orotic acid and orotidine excretion and a corresponding decrease in uridine and other purines which may have contributed to undesirable side effects. The carbohydrate analyzer has shown that there are considerable differences in excretion patterns of carbohydrates in disease. Many carbohydrates are excreted in excess in renal glycosuria and diabetes mellitus ( Y l ). Other abnormalities, such as pancreatic insufficiency and lactose deficiency, show several carbohydrate excretion abnormalities. The presence of large amounts of xylulose and other sugars during ingestion of xylose indicates that the xylose tolerance test may not be a true measurement of absorption since that sugar apparently also metabolizes ( Y l ) . Many other useful results have been found with high-resolution systems, and many of these have been reported in the previously mentioned symposia series (54, S6, S S ) . 5.

Utility and Future of High-Resolution Analytical Systems

What would one expect to gain from being able to analyze body fluids for their molecular constituents and, thus, obtain the chemical spectrum of the body? Obviously, in a more restricted sense this same question must be answered when any clinical laboratory test is being considered. If a single chemical constituent is being evaluated, its direct biochemical relationship may indicate the malfunction of a vital organ, the deficiency of an enzymatic system, the effect of a drug or other therapy, hormonal imbalance, etc. There are also pathologies where analyses for more than one constituent will allow a much better differentiation of a specific abnormality. High-resolution analytical techniques will be useful in both cases; and since a much larger number of variables will be measured, many abnormalities can be considered a t the same time. Conceivably then, the vast amount of data available from such analyses would give the clinician an additional, extremely useful tool. This will probably be the case sometime in the future; but, as the systems are being developed, there are some very real problems associated with their actual utility.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

37

5.1. DATA PROCESSING The very fact that so many data are available makes it much more difficult for the clinician to use the data since he would have to have the time and background to properly evaluate this vast amount of additional information. It would not be useful to simply give the clinician the resulting graphical chromatogram and expect him to identify and quantify the results. It would also be very time consuming to manually quantitate all of the chromatographic data by graphical separation of the resulting chromatographic peaks, finding their areas, and equating them to the concentration of each separated component. Obviously, the data can be presented in a much simpler form with the addition of computerized data reduction. The advent of relatively inexpensive, small, digital computers has made it possible to automatically evaluate the chromatographic data on-line as they are formed and end up with a tabulation of the quantity of constituents a t the end of the run ( C l , S7). It would also be a relatively simple matter to direct the attention of the clinician to those components that are outside normal limits.

5.2. CLINICALSIGNIFICANCE

At the present time, the clinical significance of an additional 300 or more body fluid constituents is not fully known and, thus, not totally useful to the clinician, although many of these components have been investigated in a rather restricted research mode. This problem will improve as additional analytical systems come into general use and additional pathological states are investigated. As indicated in a preceding portion of this paper, several interesting and useful findings have resulted from use of high-resolution systems, and these undoubtedly will continue. 5.3. ECONOMICS OF HIGH-RESOLUTION ANALYSES Will high-resolution analytical systems ever be economically feasible for large-scale use or will the cost and analysis time always be too excessive? These are important factors since analysis time for some systems may be as long as 65 hours and the cost may be as high a t $100 per sample. Although these conditions may be acceptable in the research laboratory, they could not be used on a routine basis in the clinical laboratory. Here again much progress has already been made. For example, the first prototype model of the UV-analyzer had an analysis time of about 40 hours, but recent work has decreased that time to 16 hours (S9).

EFFLUENT REDUCED TO 3.0ml BY VACUUM EVAPORATION

3 0.3

RUN TIME (hr

RUN TIME (hr) 0

50 1 0 0 1 5 0

xx)

250 300 360 400 450500

600

700

800

900

ELUTION VOLUME (ml)

1000

no0

1200

1300

1400

1500

FIQ.21. Comparison between some of the molecular constituents of human urine and the effluent streams of the primary and secondary stages of L typical municipal sewage plant as determined by the Mark I1 UV-analyzer.

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

39

Another means of economy is the analysis of multiple samples with parallel columns in a single analyzer to given an additional sample throughput of at least eight times greater than a t present (P2). Additional development work will make the economic picture even more favorable. It should be remembered that a few short years ago it took over a day to perform a semiautomated amino acid analysis on a protein hydrolysate, whereas it can now be performed in a highly automated way in less than 2 hours (Bl). We may even reach the point where i t will be less expensive, faster, and more accurate to make a high-resolution analysis of a body fluid sample even when we are interested only in a few of the constituents. This has certainly been true for the somewhat analogous case of trace metal analysis by the newer spectrographic methods instead of the more specific, but now less acceptable, wet chemistry methods. SCREENING LABORATORIES Finally, it is apparent that the field of medicine is moving toward the acceptance of and active pursuance of preventive medicine. The use of multiphasic screening laboratories is becoming more widespread to achieve this end. I n such programs, the aim is to detect incipient diseases so they can be treated prior to the need for expensive hospitalization. So far, many of the tests and techniques used in these facilities have been adapted from the clinical laboratory that operates in the hospital where acutely sick patients are present. It is obvious that as we gain more knowledge of the chemical indicators of disease, tests that are more definitive for these indicators must be developed. High-resolution systems seem uniquely suitable to such a task as they become truly economic systems. 5.4.

5.5. OTHERUSES Uses of high-resolution analytical systems in other types of research can also be envisioned. For example, the molecular pollutants, especially the refractory organic compounds, in the effluents of sanitary sewage plants have not been well established. Preliminary results from analysis of primary and secondary effluents from conventional sanitary sewage plants show that up to 80 UV-absorbing constituents can be monitored by the UV-analyzer (Fig. 21). Obviously, such analytical systems would be useful in monitoring the effectiveness of various processing steps.

REFERENCES A l . Anderson, N. G., Green, J. G., Barber, M. L., and Ladd, F. C., Analytical techniques for cell fractions. 111. Nucleotides and related compounds. Anal. Biochem. 6, 153-169 (1963).

40

CHARLES D. SCOTT

B1. Bio-Rad Laboratories, “Price L i t U. Ion Exchange Resins and Systems,” p. 20, Richmond, Calif., 1969. B2. Butts, W. C., and Jolley, R. L., Gas-chromatographic identification of urinary carbohydrates isolated by anionexchange chromatography. Clin. C h . 16, 722725 (1970). C1. Chilcote, D. D., and Mrochek, J. E., Use of automatic digital data acquisition and on-line computer analysis in high-resolution liquid chromatography. Clin. C h m . 17, 751-756 (1971). C2. Cohn, W. E., The separation of nucleic acid derivatives by chromatography on ion exchange columns.I n “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, pp. 211-241. Academic Press, New York, 1955. El. Ertingshausen, G., and Adler, H. J., Fully-automated accelerated ion exchange chromatography of amino acids in physiologic fluids. Amer. J. Clin. Pathol. 53, 680-687 (1970). H1. Hamilton, P. B., Ion exchange chromatography of amino acids. A single column, high resolving, fully automated procedure. Anal. C h m . 35,2055-2064 (1963). H2. Hamilton, P. B., Bogue, D. C., and Anderson, R. A., Ion exchange chromatography of amino acids. Analysis of diffusion (mass transfer) mechanisms. Anal. C h m . 32, 1782-1792 (1960). H3. Hamilton, P B., The ion exchange chromatography of urine amino acids: Resolution of the ninhydrin positive constituents by different chromatographicprocedures. In “Handbook of Biochemistry. Selected Data for Molecular Biology” (H. A. Sober), pp. B43-B55. Chem. Rubber Publ. Co., Cleveland, Ohio, 1968. J1. Jolley, R. L., Pitt, W. W., and Scott, C. D., Nonpulsing reagent metering for continuous colorimetric detection systems. Anal. Biochem. 28, 300-306 (1969). 52. Jolley, R. L., Warren, K. S., Scott, C. D., Jainchill, J. L., and Freeman, M. L., Carbohydrates in normal urine and blood serum as determined by high resolution column chromatography. Amer. J. Clin. Pathol. 53, 793-802 (1970). 53. Jolley, R. L., and Scott, C. D., Preliminary results from high-resolution analyses of ultraviolet-absorbing and carbohydrate constituents in several pathologic body fluids. Clin. Chem. 16, 687-896 (1970). K1. Kata, S., Confer, A., Scott, C. D., Burtis, C. A., Freeman, M., Jolley, R. L., Lee, N., McKee, S. A., Maryanoff, B. E., Pitt, W. W., and Warren, K. S., An annotated bibliography of low-molecular-weight constituents of human urine. ORNL-TM2394. U.S.At. Energy Comm., Rep. Oak Ridge, Tennwee, 1968. K2. Katz, S., Dinsmore, S. R., and Pitt, W. W., A small, automated high-resolution analyzer for determination of carbohydrates in body fluids. Clin.Chem. 17,731-734 (1971). K3. Kelley, W. N., and Wyngaarden, J. B., Effect of dietary purine restriction, allopurinol, and oxipurinol on urinary excretion of ultraviolet-absorbing compounds. Clin. Chem. 16, 707-713 (1970). M1. Moore, S., and Stein, W. H., Chromatography of amino acids on sulfonated polystyrene resins. J . Biol. Chem. 192, 663-681 (1951). M2. Mrochek, J. E., Butts, W.C., Rainey, W. T., and Burtis, C. A., Separation and identification of urinary constituents by use of multiple-analytical techniques. Clin. C h . 17,72-77 (1971). M3. Mroohek, J. E., Unpublished data. Oak Ridge Nat. Lab., Oak Ridge, Tennessee, 1971. P1. Pitt, W. W., Scott, C. D., Johnson, W. F., and Jones, G., A bench-top, automated, high-resolution analyzer for ultraviolet absorbing constituents of body fluids. Clin. Chem. 16, 657-661 (1970).

ANALYSIS BY LIQUID COLUMN CHROMATOGRAPHY

41

P2. Pitt, W. W., Unpublished data. Oak Ridge Nat. Lab., Oak Ridge, Tennessee, 1971. R1. Rosevear, J. W., Pfaff, K. J., and Moffitt, E. A., High-resolution chromatographic system for measuring organic acids in biological samples. Clin. Chem. 17, 721-730 (1971). S1. Scott, C. D., Attrill, J. E., and Anderson, N. G., Automatic, high-resolution analysis of urine for its ultraviolet-absorbing constituents. Proc. SOC.Exp. Biol. Med. 125, 181-184 (1967). S2. Scott, C. D., Johnson, W. F., and Walker, V. E., A sample injection valve for use in high-pressure liquid chromatography. Anal. Biochem. 32, 182-184 (1969). 53. Scott, C. D., and Lee, N. E., Dynamic packing of ion-exchange chromatographic columns. J . Chromatogr. 42, 263-265 (1969). S4. Scott, C. D., and Melville, R. S., co-ch., Proceedings of the first annual symposium on high-resolution analyses in the clinical laboratory. Amer. J . Clin. Pathot. 53, 677-810 (1970). S5. Scott, C. D., Jolley, R. L., Pitt, W. W., and Johnson, W. F., Prototype systems for the automated, high-resolution analyses of UV-absorbing constituents and carbohydrates in body fluids. Amer. J . Clin. Pathol. 53, 701-712 (1970). S6. Scott, C. D., and Melville, R. S., co-ch., Proceedings of the second annual symposium on high-resolution analyses in the clinical laboratory. Clin. Chem. 16, 623725 (1970). 87. Scott, C. D., Chilcote, D. D., and Pitt, W. W., Method for resolving and measuring overlapping chromatographic peaks by use of an on-line computer with limited storage capacity. Clin. Chem. 16, 637-642 (1970). 58. Scott, C. D., and Melville, R. S., co-ch., Proceedings of the third annual symposium on high-resolution analyses and advanced concepts for the clinical laboratory. C2in. C h m . 17,685-821 (1971). S9. Scott, C. D., and Chilcote, D. D., Coupled anion and cation exchange chromatography of complex biochemical mixtures. Anal. Chem. 43, 85-89 (1971). SlO. Street, H. W., The use of gas-liquid chromatography in clinical chemistry. Advan. Clin. Chem. 12, 217-309 (1969). S11. Scott, C. D. Practice of ion-exchange chromatography. I n “Modern Practice of Liquid Chromatography” (J. J. Kirkland, ed.), p. 313. Wiley, New York, 1971. 512. Scott, C. D. Analysis of urine for its ultraviolet-absorbing constituents by highpressure anion-exchange chromatograph. Clin. C h m . 14, 521 (1968). T1. Thacker, L. H., Scott, C. D., and Pitt, W. W., A miniaturized ultraviolet flow photometer for use in liquid chromatographic systems. J . Chromatogr. 51, 175-181 (1970). T2. Thacker, L. H., Pitt, W. W., Katz, S., and Scott, C. D., Miniature photometers for liquid chromatography. Clin. Chem. 16, 626-632 (1970). Y1. Young, D. S., High-pressure column chromatography of carbohydrab in the clinical laboratory. Amer. J . Ctin. Pathol. 53, 803-810 (1970). Y2. Young, D. S., Epley, J. A., and Goldman, P., Influence of a chemically defined diet on the composition of serum and urine. Clin. Chem. 17, 765-773 (1971).