Automated analyses of sugar phosphates

ANALYTIC4L

BJOCHE.MISTRY

Automated

9, 293-302

(1964)

Analyses

of Sugar

Phosphates

J. R. BURT From the Department of Scientific and Industrial Research, Tovy Research Station, Aberdeen, Scotland Received February

17, 1964

INTRODUCTION

The methods of analysis for sugar phosphates hitherto used in this laboratory (1) are tedious to perform since they involve the carrying out of up to 100 colorimet.ric determinations per sample analyzed. The possibility of adapting the AutoAnalyzer system to these analyses was investigated and this paper describes procedures for determining hexose and pentose phosphates in fractions collected after ion-exchange chromatography and also for the direct determination of hexose phosphates by continuous monitoring of the effluent. from an ion-exchange column. The ion-exchange procedure previously described (1) has been modified slightly to allow for a more rapid separation of sugar phosphates. Syamananda, Staples, and Block (2) have published details of a method for determining sugars in effluents from an ion-exchange column with anthrone and sulfuric acid using an AutoAnalyzer and the monitoring procedure described here is basically similar. The anthrone method was chosen since it reacts with all hexosesand does not require a preliminary hydrolysis of the hexose phosphates [see ref. (1) 1. For the same reasons, the orcinol reagent of Mejbaum (3) was chosen for the automation of pentose phosphate determinations. METHODS

Ion Exchange The ion-exchange procedure described previously (1) for the separat,ion of sugar phosphates has been modified slightly to obtain a complete separation within 6 hr. The grade of resin and concentrations of eluting solutions remain t.he same, but the dimensions of the column are reduced to 28 cm X 1.6 cm and the volume of the mixing vessel is reduced from 1 1. to 750 ml. The total time t,aken to complete the elution of fructose diphosphate is under 5 hr and the total effluent volume to that point is approximately 750 ml. 293

294

J.

(‘ontinuows

R.

BURT

Monitoring

with

dnthmne

For the continuous analysis of the column effluent an AutoAnalyzer’ consisting of proportioning pump, heating bath wit.h two coils, calorimeter and recorder is used. The flow diagram is illustrated in Fig. 1. Acidflexl tubing is used for all lines carrying sulfuric acid. The column effluent is split into two streams at A, the larger one being collected in approximately lo-ml fractions. The smaller stream (about 10% of the total column effluent) is segmented with air B, cooled C, and blended D with a stream of 0.1% anthrone in 76% sulfuric acid (made fresh daily)

FIG. 1. Flow diagram for hexose and hesose phosphate toring with anthrone reagent.

analysis. Continuous

moni-

Rowing at 5 times the rate of the sample. The T-piece is fixed vertically in the cooling bath so that reagent enters the segmented sample stream from below. After mixing in coil E, the reaction mixture passes through the heating bath where its dwell-time in the single coil is approximately 13 min with the flow rates specified. The stream is then cooled and passed into an S-mm flow cell in the calorimeter where optical densities are recorded at 618 mp. Before connecting the AutoAnalyzer to the ion-exchange column it is ’ Technicon

Instruments

Co. Ltd., Chertsey, Surrey, England.

essential to prime the system by running the instrument with water flowing through the sample line and 76% sulfuric acid flowing through the reagent line. After a IO-min running under thcsc conditions, the reagent line can be connected to the stock of anthrone reagent, and after a furt’her 15 min the sample line is connected to a stock of standard sugar solution so that a plateau, which can be used for calibration, is obtained in the record. The recorder baseline is set when a steady level has been reached with the blank reagent mixture flowing through the colorimetcr. After a 20-min running with the standard the sample line is cleared with water again and then connected t.o the column outlet. While the AutoAnalyzer is being set up and calibrated, elution of the column is started. The first 150 ml of effluent can be discarded, as no compounds of interest are contained therein. All changes of tubing from one solution to another should be carried out’ as rapidly as possible so that the minimum amount of air is introduced into the sample and reagent lines. The reason for this is discussed later. Careful positioning of the sample stream splitter (A) ensures that any air introduced prior to that point enters the limb leading to the fraction collector and that none enters the analytical stream. Sampling

Procedure

with

Anthrone

Using the sampler’ module fitted with a double crook, in addition to the modules used for continuous monitoring, hexose or hexose phosphate concentrations in individual samples as well as in a series of fractions collected after column chromatography can be determined. The flow diagram for this procedure is shown in Fig. 2. The sampler is run at 40 samples per hour. The times taken for liquids to reach point A should be as near as possible the same in each path from the double crook. The “debubbler” T-piece B should have a, very short length of tubing connecting it to the pump and thence to C, where the sample stream is resegmented with air. The arrangement of the system from C onward is identical with that in the continuous monitoring system except that the heating bat.11now uses both coils. Again, Acidflex tubing is used for all lines carrying sulfuric acid; the composition of the anthrone reagent is the same as above; and the dwell-time in the heating bath is 12.5 min. The preliminary procedure is similar to that in the monitoring system. The AutoAnalyzer is run with both lines from t,he double crook in water and the reagent line in 76% sulfuric acid for 10 min. The reagent line is then transferred rapidly to the stock of anthrone reagent and the system run for a further 15 min before t.he sampler is started. The baseline is set on the recorder when a steady optical density level has been attained with reagent flowing through the calorimeter.

FIG. 2. Flow diagram for hexose and hexose phosphate dure with anthrone reagent.

analysis. Sampling

proce-

Sampling Procedure with Orcinol This procedure is a straightforward automation of the manual method of Mejbaum (3). The flow diagram is shown in Fig. 3. The orcinol reagent consists of 1% orcinol freshly dissolved in concentrated hydrochloric acid containing 0.1% ferric chloride. The dwell-time in the heating bath is approximately 15 min, and the sampler is run at, 40 samples per hour. RESULTS

AND DISCUSSION

Sample separations of glucose l-phosphate, glucose 6-phosphate, fructose l-phosphate, fructose 6-phosphate, and fructose diphosphate are shown in Fig. 4 as a result of continuously monitoring the effluents from ion-exchange columns. The amounts of hexose corresponding to each peak are calculated by using the formula: X = WHfc/SMh where X = apparent pmoles hexose phosphate in peak W = width (in.) of peak at half-maximum optical density H = optical density of peak maximum

S f c M

= = = =

chart speed (in/mm) flow rate (ml/min) from column concentration of standard (pg/ml) to give optical density molecular weight, of standard compound

of h

It was found that, under the conditions operating in this dutoAnalyzer system, there is not a molar equivalence among glucose and the various hexose phosphates investigated so that the value S, in cases in which glucose standards are referred to, is only the apparent glucose equivalent of the sugar phosphate in any particular peak. However, if a standard mixture of sugar phosphates is separated by ion-exchange chromatography and analyzed by this system the S value corresponding to each peak can be used as a standard t,o which peaks in other runs can be referred, provided that t.he same compound is run as a standard in each case and that Beer’s Law is still operat,ing. The concentration of glucose is proportional to the optical density produced in this method at least up to 150 pg/ml. Table 1 shows the recoveries of standard mixtures of hexose phosphates at different concent,rations in a series of five experiments. Reasonable recoveries were obtained except for experiment (ii) in which the two fructose monophosphates were run together. The partial separation (see Fig. 4) of these twc, compounds makes it impossible to be certain of the validity of any calculation of concentrations. A peak containing 0.05 pmoles of hexose phosphate can be detected

FIG.

cedure

3. Flow diagram for with orrinol reagent,.

pentose

and

pentose

phosphate

analysis.

Sampling

pro-

14.06 9.36

4.68

4.6s

(ii) (iii)

(iv)

(v)

SCStandard

14.06

run.

/Lmoles

(i)

Expt.

6.35

5.70

17.7G 9.50

li.78

1Oi

9G

100 80

100”

II.68

7.00

1.67

7.00

7.00

/moles

15.20

8.80

10.04 5.75

9.34

98

94

111 93

100”

2.59

7.55

7.55

pmoles

1

2.90

15.78

8.14

107

194

100”

% recovery

l-phosphate

details)

ANALYSIS

X Wdlle

Fructose

TABLE BY CONTINUOUS (see test for

% recovery

X VrtlWS

% recovery

Glucoac G-phosphate

PHORPH.\TES

X V&le

OF HEMXE

Glucose l-phosphate

RECOVERIES

12.20 s.14

#moles

COLUMN

4.W

4.20

100”

79

% reco”ePy

6-phosphate

X value

Fructose

OF ION-EXCH.\NGE

9.2G

G.95 4. u.3

G.95

pmoles

13.3

12.20 G.80

IO.67

93

I14 9G

100~~

% recovery

diphosphate x VrtlUe

Fructose

EFFLUENT

z 3

.?

;i

FIG. 4. Chromatograms of hesose phosphates obtained by treating effluent from ion-exchange column continuously with anthrone reagent according to flow diagram in Fig. 1. Plateaux A obtained from 100 .ug glucose per ml; peaks B, C, D, E, F are glucose l-phosphate, glucose 6-phosphate, fructose l-phosphate, fructose B-phosphate, fructose diphosphate, respectively. Concentrations of hexose phosphates in (a) and (b) above are listed in experiments (i) and (ii), respectively, in Table 1. Chart speed, 0.0597 in./min.

readily provided neighboring peaks are not disproportionately wide. The upper limit of usefulness is over 40 pmoles per peak although at this level chromatographic resolution may have suffered. The insolubility of anthrone in water renders it essential to run 76% sulfuric acid through the reagent lines before t,hey are connected to the stock of the reagent. If this procedure is omitted there is a real danger of anthrone’s being precipitated and a blockage occurring. This caution applies also to the anthrone-sampling procedure, which also introduces other problems. Preliminary experiments using a single crook at the sampler and omitting the recycling of the sample (Fig. 2A-C) gave extremely (‘noisy” recordings, which were clue to the fact that, during sampling, the sulfuric acid in the reagent was diluted to 63% while, during the changeover between samples, air was being aspirated through the sample line and the sulfuric acid concentration remained at 76%. When the mixing of these two solutions of different strengths of sulfuric acid takes place in t’he flow cell, striae which move across the light path

300

.I. a. UlrHT

are produced, as the refractive indices of the two solutions are very different. As each stria occludes and then clears, a sharp kick is traced on the recording. Introduction of the double crook with an intersample wash cleaned up the tracing a lot, but as it was impossible to arrange that the front of the intersample wash would reach point A (Fig. 2) just as the back of the preceding sample did, and that the front of the succeeding sample would coincide at that point with the back of the intersample wash, occasional kicks still appeared on the recording. Recycling the sample line as illustrated in Fig. 2 ensures that line BC always contains water and that the reaction mixture flowing through the calorimeter has a constant sulfuric acid and water composition. Despite these precaut,ions, relatively poor intersample wash characteristics, due to the high viscosity of the reagent, are evident in records obtained from the anthrone-sampling procedure, and appreciable contamination of a sample by the one preceding also occurs. When running unknowns through this system, it is therefore advisable, if the samples are at random, to run them in triplicate and to ignore the first two values obtained. However, if concentrations of succeeding samples are known to be similar or to change only gradually as in fractions collected after ion-exchange chromatography it is necessary to run these samples only once. For calibration, a standard graph is prepared from standard glucose solutions, containing up to 250 pg/ml, run in triplicate. A linear plot which passes through the origin is obtained when the optical densities (corrected for the blank) of the third sample at each concentration are plotted against concentration. As little as 2 pg glucose/ml can be detected, and linearity is lost at. a little above the 250 pg/ml level. Normally it is necessary to run a standard, consisting of a single concentration only, four times in immediate succession every 2 hr or so, provided that this standard and all unknown samples are within the linear portion of the color response. As in the continuous monitoring procedure it was found that molar equivalence of color response among glucose, fructose, and various hexose phosphates was not obtained in the sampling procedure. Table 2 gives the optical densities of various hexose phosphates in the sampling procedure described here relative to glucose at equal molecular concentrations. Consequently, apparent concentrations of samples calculated from a glucose standard should be divided by the figures contained in Table 2 in order to arrive at a true value. In a series of three experiments in which standard mixtures of hexose phosphates of varying concentrations were resolved by ion-exchange

TABLE GLCCOSE

EQUIVALENCES

0.05 0.86 1.11 0 !14 1.40 1.26

l-Phosphate B-Phosphate l-Phosphate &phosphate diphosphate

PROCEDURE Standard error of mean

Mean

Sugar

Fructose Glucose Glucose Fructose Fructose Fructose

2

IN AE~THRONE-HAMPIJNG

0.040 0.049 0, O-l!) 0.069 0.04!) 0.040

chromatography into fractions which were subsequently analyzed for hexose content by this sampling procedure, the percentage recovery was found to be 85 and 104 for glucose l-phosphate; 84 and 103 for glucose 6-phosphate; 103 for fructose l-phosphate; 105 for fructose 6-phosphate; 98 and 108 for fructose diphosphate, taking the first cxpcriment as the basis on which these recoveries were calculated. The determination of pentose with orcinol in the AutoAnalyzer has turned out to be a compromise. In order to obtain a reasonable time for color development, the volumes of sample and reagent were kept comparatively low and this brought about rather sharp sample peaks, although it was found that Beer’s Law is observed at least up to 30 pg ribose/ml. As it is, the 15-min dwell-time in the heating bath at 95°C is considerably less than the optimal 45 min at loo”, by which t,ime all pent,oses (free and combined) will have developed equivalent, maximum colors (4). Since the rates of color development in this reaction are different for various pentoses and pentose phosphates (4), widely different equivalent color responsescan be expected, depending on the nature of the pentose tested. This method has not been explored any further than this, but it would be simple to determine conversion factors (analogous to those found for various hexose phosphates in the automated anthrone reaction) for calculating concentrations from ribose (or any other pentose) standards. ACKNOWLEDGMENTS The author wishes to thank Mr. W. H. C. Shavv of Glaxo Laboratories Ltd.. Greenford, England, for some useful suggestions which have been incorporated in the anthrone sampling procedure. The work described in this paper was carried out as part of the program of the Department of Scientific and Industrial Research. Crown

Copyright

Reserved

302

-1. R.

BURT

REFERENCES

L. Josm,

N. R.,

AXD BURT, J. R., Analyst 85, 810 (1960). R., STAPLES, R. C., AND BLOCK, R. J., Contrih. Boyce Thompsc Inst. 21, 363 (1962). 1. MEJBNJM, W., 2. physiol. Chem. 258, 117 (1939). 1. ALBAUM, H. G., AND UMBREIT, W. W., J. &ok Chem. 167, 369 (1947). !. SYAMANASDA,