thin-layerchromatographyof urinary carbohydrates

954 CLINICAL CHEMISTRY, Vol. 16, No. 11, 1970

Thin-LayerChromatographyof Urinary CarbohydratesA Comparative Evaluation of Procedures

D. S. Young and Agathe J. Jackson

Ten procedures for thin-layer chromatography of carbohydrates in urinewere compared. The procedures differ considerably in their sensitivity andability to separate sugars of clinical importance. A procedure was developedthat permitted several sugars to be identified in normal urine samples with-out prior desalting or concentration. Celite was used as adsorbent, anisalde-hyde as location reagent. The procedure is simple and reproducible, and isreadily modifed for quantification of sugars by thin-layer densitometry.

Additional Keyphrases sugars in normal and pancreatic disease urine #{149} 10TLC systems compared #{149} semiquant ifi cation #{149} Celite adsorbent, anisaldehydespray results by column chromatography compared

SEVEItAL METABOLIC disorders are characterizedby the excretion of abnormally large quantities

of sugar in the urine. It has been recognized onlyrecently that normal urine contains many sugarsat a low concentration, the clinical significance ofwhich has not yet been established (1). TLC1 tech-niques are presently available for detecting sugarsin abnormal samples, but prior concentration ofsamples is required to detect the quantity of sugarsnormally present in urine. To determine the clini-cal significance of small differences in the usualexcretion pattern of carbohydrates it is necessaryto develop a screening technique sensitive enoughto detect small concentrations of the commonurinary sugars.

The numerous samples that a clinical laboratorymay be required to examine necessitate use ofunidimensional chromatography without priordesalting or concentration of samples. In this

From the Clinical Pathology i)epartment, Clinical Center,National Institutesof Health, Bethesda, Md. 20014.

This paper was reviewed before submission. Received and ac-cepted Sept. 10, 1970.

1 Abbreviation used: TLC, thin-layer chromatography(-graphic).

study, we compare the separation of carbohy-drates by several different TLC procedures (notnecessarily originally proposed for physiologicalfluids) with a technique that we have modifiedfrom that of Garbutt (2), which we found to bemost suitable for identifying and estimating car-bohydrates in urine. The different systems wereevaluated for their ability to adequately separateurinary sugars in a single dimension with minimumsample preparation. The ease with which theycould be measured by thin-layer densitometry wasalso evaluated, as was the reproducibility of theseparation and interference from noncarbohydrateconstituents of urine.

Materials and Methods

Reference System

Glass plates. Plates (20 X 20 cm) were preparedwith a Desaga apparatus (C. Desaga GmbH,P.O. Box 407, Heidelberg, West Germany). Theadsorbent was Filter-Cd (Celite, Johns-MansvilleCo., Celite Division, New York, N.Y.), spread toa thickness of 250 u. For the preparation of fiveplates, 15 g of Celite was ground with 2 g of an-

Adsorbent

Table 1. Characteristics of Evaluated TLC Systems

CLINICAL CHEMISTRY, Vol. 16, No. 11, 1970 955

System Reference

A Reference System

B Garbutt(2)

C Eastman Kodak

D Eastman Kodak

E Anderson andStoddart (4)

F Hay #{128}1at. (s)

G Lato et at. (6)

H Adachi (7)Paget and

Coustenoble (8)J Wolf rom et al. (9)

Celite

Celite

Silica-gel Chromagramwith sodium bisulfite

Silica-gelChromagramwith sodium acetate

Silica-gel Chromagram

Silica gel

Silica gel

Silica gel

Silica gel

Cellulose MN 300

Solvent (proportions by volume)

Ethyl acetate: pyridine:water(60:25:20)

n-Butanol: pyridine:water(75:15:10)

Ethyl acetate: methanol:aceticacid:water (60:15:15:10)

Acetone: chloroform: methanol:water (80:10:10:5)

Butanone :acetic acid: water(3:1:1)

1-Butanol :acetic acid: ethylether:water (9:6:3:1)

n.Butanol:ethyl acetate: isopro-panol:acetic acid:water(35:100:65:35:30)

n-Propanol:water (17:3)n-Butanol: methanol: water

(5:3:1)Ethyl acetate: pyridine:water

(2:1:2) (upper phase)

Location reagent

Anisaldehyde/H,S04

Ammoniacal AgNO,

Aniline hydrogenphthalate/CH,COOH


Naphthoresorcinol/H,S04

Resorcinol in phosphoricacid/H2S04

Naphthoresorcinol/H,504

Thymol/H,S04Thymol/H,504


hydrous CaSO4 in a mortar. Aqueous sodium ace-tate, 60 ml of 20 mmol/liter solution., was addedand mixed for 1 mm in a Waring Blendor. Theplates (now commercially available from AnaltechInc., Wilmington, Del.) were poured immediately,dried with a hot-air fan, and stored under reducedpressure in a dessicator. They were activated be-fore use by heating at 100#{176}Cfor 30 mm.

Marker solution. D-Glucuronic acid, lactose,maltose, sucrose, D-galactose, D-glucose, D-fruc-tose, D-mannose, L-arabinose, D-ribose, and D-xylose, 100 mg of each, were dissolved in 100 ml ofwater. The mixture was stored in small aliquotsat -20#{176}C.

Quantity of urine to be chromato graphed. Becausethe concentration of salts has a considerable in-fluence on Rf values of sugars, the volume of urineapplied to the plate was determined by the specificconductivity of the sample, as measured on aconductivity bridge, Model 31 (Yellow SpringsInstrument Co., Yellow Springs, Ohio). Differentvolumes were applied to the plate according to theschedule: specific conductance greater than 22mmhos/cm, 2.5 of urine was applied to the plate;between 15 and 22 mmhos/cm, 5.0 1d; and be-tween 10 and 15 mmhos/cm, 12 zl. Samples wereapplied as streaks 1-cm long and 1-cm apart. Amicrotitrator (Beckman Instruments Inc., Fuller-ton, Calif.) was used to apply the sample.

Solvent. A mixture of ethyl acetate, pyridine,and water (60:25:20, by volume) was preparedfreshly for each migration. The time required for10-cm migration in “sandwich” chambers (Brink-mann Instruments, Inc., Westbury, N.Y.) wasabout 75 mm. After the solvent had migrated 10cm, the plates were dried with a cool-air fan until

the smell of pyridine had disappeared. Althoughseparation by a single migration was adequate, asecond migration with fresh solvent improved theresolution of the sugars.

Location reagents. For detection of the sugars,the dried plates were sprayed with about 5 ml ofa mixture of 9 ml of absolute ethanol, 0.5 ml ofconcentrated sulfuric acid, and 0.5 ml of anisalde-hyde. The plates were then heated at 100#{176}Cuntilthe color of the spots was maximum (20 to 30 mm).

For measurement of the sugars, p-aminoben-zoic acid was prepared as described by Saini (3)and used as a spray. Reddish-brown spots wereproduced with all carbohydrates when the plateswere heated to 105#{176}to 110#{176}C.The sensitivity ofthis reagent was comparable to that of anisalde-hyde. The sugars were estimated by scanning theplates in a Spectrodensitometer (Farrand Optical,Mt. Vernon, N.Y.) at 550 nm.

If the excretion of a sugar appeared to be ab-normally high, the stock sugar was diluted withwater, to contain 0.5 and 5.0 /.Lg/J.Ll. These wereapplied as streaks of 10 The absorbance of thesugar in the urine sample could be compared withthat of the standard solutions to gain a semiquan-titative estimate of its concentration.

Evaluation Procedure

Nine different systems as described in the litera-ture were compared with the Reference System(Table 1). The procedures were followed as recom-mended except that Brinkmann sandwich cham-bers were used throughout for all plates andChromagram sheets (Eastman Kodak Co.,Rochester, N.Y.). The same system was run induplicate on three different days and the same

Table 2. Operating Conditions of Different TLC Systems for Sugars, as Required for OptimumSeparation and Sensitivity of Sugars

System’ A B C D E F G H

See Table 1.

Migration distance, cm 10 12 10 10 12 12 12 13 12 12Development time, mm 75 x 2 280 70 35 80 165 215 295 100 25 x 2Thickness of adsorbent, z 250 250 100 100 100 200 300 250 300 500Volume marker solution, l 3.0 3.0 3.0 3.0 10.0 3.0 5.0 5.0 3.0 10.0Volume undesalted urine, Ll 2.5 2.5 2.5 5.0 10.0 5.0 10.0 5.0 5.0 10.0

J

46RBr!40 RBr!34Y!

32BrY!31 BrY!

13 Y!

59 GyP 25GyB 63R29YBr!21 YBr!16YBr!

14 Br!llBr!

4 Y!

52 B47 B46843 R41 B39 B34 R25B228

Carbohydrate A B C D E F G H I J

Ribose 71 GyG 51 V 52 RBr! 30 YBr! 54 B 43 RP 64 BXylose 70fYG 73VArabinose 58Y 58VMannose 53YG 63VFructose 52B 55VGlucose 46 B 62 VGalactose 39 G 52 VSucrose 31 B 45 fVMaltose 27 B 36 VLactose 19 GB 22 VGlucuronicacid 8 Pk 0 V 19Y! 0 BrY! 28 B 11 RP 36 BV 21 fBr 9 Gy 12Or

valuesare Rf values X 100 and represent the mean of duplicates on three different days. Color reactions not visible with loca-tion reagent; d, dark; f, faint; !, fluorescent; Pk, pink; G, green; B, blue; Y, yellow; Gy, grey; Br, brown; R, red; P. purple; v, violet; Or.orange.

56 RP43 RP44 RP36 RP41 RP34 RP30 RP24 RP16 RP

63 B54 B55 B52 R52 GyV45 GyV41 R35 Pk26V

65fP54 P58Br56 P55 P49 P52 P47 P34 P

48 GyB52 GyB50 Br20 Pk50 Pk42 Pk61 Pk58 Pk41 Pk

53R47 R46dG

4idG36dG

29fPk23dG


samples were run on each occasion. The sampleswere:

#{149}3 zl of the 11-sugar “marker” solution listedabove.

#{149}3,.d of five “clinically important” sugars(glucose, lactose, galactose, sucrose, and fructose)in water.

#{149}Urine I, from a normal subject (with specificconductance 25.7 mmhos/cm).

#{149}Urine II, from a patient with pancreatic dis-ease, containing several sugars in abnormal con-centration (specific conductance, 20.6 mmhos/cm).

The two urine samples were applied withoutdesalting. Individual sugars in water were run toconfirm the identity of sugars in the mixtures ifthe sugars did not yield characteristic colors, or ifseparation was inadequate.

Three additional systems (10, 11, 12) were in-vestigated but found to be unsatisfactory (seebelow). All systems were used according to thepublished description. If the location reagent wasunavailable or unsatisfactory, other location re-agents were evaluated. The ambient temperatureduring all experiments varied between 24.5#{176}and27#{176}C.

System J (Table 1) was run both in sandwichchambers and a tank. Only the Rf values fromruns in a tank are reported because the solventseparated into two phases in the sandwich cham-bers, and the spots were less well resolved.

Results

Main differences between the operating condi-tions of the 10 systems are summarized in Table 2.Because of the different sensitivities of the locationreagents, volumes of marker solutions and urinewere adjusted to ensure reasonable clarity of spots.Rf values of the sugars in the 11-sugar markersolution are listed in Table 3, and represent themean value for duplicate runs on three differentdays. The color of the sugar spots with the variouslocation reagents is also included.

Separation capabilities of the different systemsare summarized in Table 4. The location reagents,or their mode of application, had to be modifiedfrom the original description for several systems asindicated below.

Comparison of Systems

System A. The 11 sugars in water separated intonine clear bands (only the resolution of ribose fromxylose, and of fructose from mannose being in-adequate). Rf values of the sugars in undesaltedurine were about 5% less than in water. However,the distinctive colors with anisaldehyde helped usidentify the sugars without difficulty. Urea gavea yellow spot, fading to white, with an Rf valueslightly greater than that of ribose. Uniformlythick plates were hard to prepare.

System B. Only five bands could be identified

Table 3. Migration Distances and Reaction with Location Reagent Compared for Systems in Table 1”System

Number of discrete bandsfrom 11-sugar mixture 9 5 0b 0 5 4 5 7 4 8

Number of discrete bandsfrom 5-sugar mixture 5 3 2 2 4 3 3 3 3 3

Additional bands in urine Inot due to referencesugars 1 1 2 2 1 1 2 1 1 1

“See Table 1.A listing of 0 indicates that a continuous streak was present

without clear differentiation of separate components.


Table 4. Apparent Separation Capabilitiesof Different Systems

System” ABCDEFGH I J

from the 11 sugars in water. Only glue uronic acid,lactose, maltose, and xylose separated as discrctebands; the other sugars remained in a continuousstreak. The five-sugar mixture separated intothree bands. Sucrose stained very weakly with therecommended location reagent (silver nitrate inammonia). Migration in sandwich chambers re-quired 4.5 h, in contrast to the 0.5 h cited by theauthor, who used a tank.

System C. Maltose, sucrose, and fructose failedto yield a visible or fluorescent spot on sprayingwith the location reagent (aniline hydrogen phtha-late in glacial acetic acid). Urine samples producedone yellow fluorescent band and one visible pinkband, which could not be correlated with anyknown sugars.

System D. The same criticisms apply to thissystem as to System C. The low Rf values of allsugars further impaired the resolution of bandsand rendered tentative identification by Rf moredifficult.

System E. The separation of the 11 sugars inwater was poor. The recommended location re-agent (ethanolic aniline oxalate) yielded yellowsmudges and protracted heating of the sheetsmelted them. The use of napthoresorcinol in eth-anol and concentrated sulphuric acid enabled fivebands to be identified from the 11-sugar markersolution, and four bands from the five-sugar solu-tion. Separation of the carbohydrates in urine waspoor. Pink and grey bands were produced by theurine samples, which could not be correlated withknown sugars.

System F. Only four bands were identifiablewhen the 11-carbohydrate solution was applied,and three were identified in the five-sugar solution.Urine specimens yielded three bands, one of whichhad a greater Rf value than any of the sugars inthe marker solutions. One of the other spots wasdue to xylose, and the third was very diffuse andcould have contained glucose, galactose, fructose,mannose, arabinose, or ribose. Sulfuric acid, usedby us as location reagent, gave faint brown spotson heating but, when resorcinol (20 g/liter) in

phosphoric acid was next applied, strong red-purple spots appeared on a yellow-pink back-ground. Glucose and fructose were better separatedin the sandwich chambers than described in theoriginal description, in which a tank was used.

System G. Five bands were separated from the 11-carbohydrate solution, three from the five-com-ponent solution. Glucose and fructose had identi-cal Rf values. With urine, additional spots ap-peared above the sugars of greatest Rf value, buttheir identities were not detemined. The adsor-bent-silica gel without binder-tended to flakeand become powdery, producing uneven solventfronts.

System H. Seven bands were separated from the11-sugar marker solutions, three from the five-sugar solution. Urine samples produced two spots;one was glucose, the other migrated further thanany marker sugar. Glucose and fructose had simi-lar Rf values. The strength of the location reagenthad to be increased over the recommended con-centration to produce strong spots.

System I. Because of streaking on the plate, onlythree distinct bands were discernible from themarker solutions. The location reagent gave weakcolors with all the sugars and galactose was poorlyseparated from lactose.

System J. Eight spots were produced from the11-sugar marker solutions, three from the five-sugar solution when a tank was used. As with sys-tems C and D, aniline hydrogen phthalate in gla-cial acetic acid failed to make fructose and sucrosevisible at the concentrations present. Sugars inurine were incompletely separated but desaltingimproved the resolution. Cellulose MN 300 (Brink-mann) was used in preference to Avicel (recom-mended by the authors) because of difficulty in pre-paring uniformly coated plates.

Other systems. Even when 15 il of urine andmarker solutions was applied to the plate in thesystem described by Becker and May (10), thespots that developed were very faint, and the car-bohydrates in the mixtures failed to separate.The system described by Kudla and McVean (11)was rejected because of the need for continuousmigration to obtain adequate separation of sugars,as the solvent front travels much more rapidlythan any of the sugars. The system did producegood separation of the sugars in both marker solu-tion and urine, although the colors produced byreaction of the location reagent with the sugarsfaded within 5 mm. Permanent colors and a well-defined solvent front are necessary requirementsof a system for identification and measurement ofsugars.

The location reagent used in the original methodof Vomhof and Tucker (12) is no longer commer-cially available. Other location reagents failed todemonstrate clearly the sugars in the referencemixture, although the same reagents had proved


System

Table 5. Carbohydrates

A B

in Two Undesalted Urines, asby Use of Different Systems”

C D E F

Provisionally

G

Identified

H I .1

Urine I D,F,GA D,GA,L,X GA GA 0 X D,GA D,X 0 GAUrine II D,F,GA,S D,F,GA,L,X GA ?D,GA D X D,G,GA D,X D,F D,X

D, glucose;F, fructose; G, galactose; GA, gl ucuronic acid; L, lactose; 5, sucrose; X, xylose.

successful with the other systems discussed pre-viously.

Compounds Detected in Urine

A tentative identification of the carbohydratesdetected in undesalted urine from normal subjectI and from patient II (with pancreatic disease), asindicated by the different systems, is listed inTable 5. There was little difference between thecompounds identified before and after desalting,although galactose and lactose were detected inurine II, when system A was used after desalting.

When urines I and II were high-pressure colunm

chromatographed according to the procedure de-scribed by Jolley and Freeman (1), many addi-tional carbohydrates were detected in both sam-ples. The column chromatographic system wasused to determine the concentration of the sugarsdetected by the thin-layer chromatographic sys-tems (Table 6). Glucuronic acid is not included, asit has not yet been possible to quantify it with thecolumn-chromatographic system.

DiscussionRequirements for a thin-layer chromatographic

system for urinary sugars differ, depending onwhether the objective is to detect one compoundthat is present in great excess or many sugarspresent in their normal concentrations. When thelatter is important essential considerations are theadequate separation of the compounds and sensi-tive location reagents. The sugars we included inthe five-component marker solution are importantin the metabolism of carbohydrates in humans.

Table 6. Approximate Concentrations of Sugars(mg/liter) in Urines I and II, as Determinedby High-Pressure Column Chromatography

Urine I Urine II Normal range (IS)

Sucrose 40 200 0-150Lactose 10 80 0-100Fructose 10 170 0-50Galactose 20 40 0Xylose 25 90 0-30Glucose 150 570 10-120

Adequate separation systems should isolate thesesugars from each other and from ribose and arabi-nose, which are also normal urine constituents.

Even with compact spots a difference of 0.03 be-tween Rf values of two sugars is necessary forclear differentiation, so that tentative identifica-tion may be assigned. System A produces adequateseparation of the important carbohydrates, al-though ribose is not well separated from xylose,nor fructose from mannose. However, the differ-ence in color produced by reaction of these com-pounds with anisaldehyde helps in identification.The other systems either failed to separate thesugars adequately (as with Systems B,E,F,G,H,and I), or the location reagent failed to react withsome of the important sugars (Systems C,D, andJ).

Location reagents such as anisaldehyde, whichyield different colors with different sugars, areuseful because both characteristic color and Rfvalue are useful in provisional identification of thecompounds. For quantification by densitometry auniformly colored spot against a light backgroundis desirable. Standards of all the sugars to be mea-sured should be included in the same run. Mes andKamm (14) have recently compared the sensitiv-ity of various location reagents for carbohydrates.Their study indicated a large difference betweensensitivities, which partly accounts for the differ-ent sugars found in the urine samples in this study(Table 5). The combination of adsorbent and loca-tion reagents used for Systems A and B allowed agreater number of sugars to be identified thanwith any other system except J, for which fourtimes the volume of sample was used.

Because different chromatographic systems andlocation reagents do not necessarily reveal thesame carbohydrates in the same sample, cautionmust be used in interpreting results. Additionalseparations with different location reagents shouldbe performed before definitive identities should beascribed to provisionally identified compounds.This study has demonstrated that it is possible toseparate several different carbohydrates from asmall volume of normal urine. This permits thedevelopment of a systematic approach for rapidscreening of urine for small variations in composi-tion of carbohydrates so that abnormal samplescan be identified for study in greater depth bymore sophisticated procedures.


References

1. Jolley, R. L., and Freeman, M. L., Automated carbohydrateanalysis of physiologic fluids.CLIN. CHEM. 14, 538 (1968).

2. Garbutt, J. L., Inexpensive adsorbents for thin-layerchroma-

tography of carbohydrates. J. Chromatogr. 15, 90 (1964).

3. Saini, A. S., Some technical improvements in the paper chro-matography of sugars. A method of sample desalting and a sensi-tive staining reagent. J. Chromatogr. 24, 484 (1966).

4. Anderson, D. M. W., and Stoddart, J. F., Separations ofsugars on “Chromagrams.” Carbohydrate Res. 1, 417 (1966).

5. Hay, G. W., Lewis, B. A., and Smith, F., Thin-film chroma-tography in the study of carbohydrates. J. Chronw.Iogr. 11, 479(1963).

6. Lato, M., Brunelli, B., Ciuffini, G., and Mezzetti, T., Bidimen-sional thin-layer chromatography of carbohydrates on silica gelimpregnated with boric acid. J. Chromoiogr. 34, 26 (1968).

7. Adachi, S., Thin-layer chromatography of carbohydrates inthe presence of bisulfite. J. Chromatogr. 17, 295 (1965).

8. Paget, M., and Coustenoble, P., La chromatographie surcouches minces des oses et osides rencontr#{233}sdans des enzymo-pathies affectant.lesglucides.Ann. Biol. Clin. 25, 1239 (1967).9. Wolfrom, M. L., De Lderkremer, R. M., and Schwab, G.,Quantitative thin-layer chromatography of sugars on micro-crystalline cellulose. J. Chromatogr. 22, 474 (1966).10. Becker, S., and May, P. Thin-layer chromatography of urinesugars. Amer. J. Gun. Pathol. 49, 436 (1968).11. Kudla, R. M., and McVean, D. E., Semiquantitat.ive separa-tion of carbohydrates in urine by thin-layer chromatography.Amer. J. Gun. Pat hot. 49, 279 (1968).

12. Vomhof, D. W., and Tucker, T. C., The separation of simplesugars by cellulose thin-layer chromatography. J. Chronwlogr. 17,300 (1965).

13. Long, C., Biochemists’ Handbook. Van Nostrand, Princeton,N.J., 1961, p 923.

14. Mes, J., and Kamm, L., The relative sensitivity of variousreagents for the detection and differentiation of sugars and sugarderivatives in glycoproteins. J. Chromatogr. 38, 120 (1968).

thin-layerchromatographyof urinary carbohydrates

Documents