V O L U M E 2 7 , NO. 2, F E B R U A R Y 1 9 5 5 Table IV. Component Propane n-Hexane Hexenes a n d / o r Cs cycloparaffins Benzene Suma
239
Determination of Hexanes Known 0.00 0.226 0.00 0.00 0.226
Mole % ' Palladium Tube-Mass Spec. 1 2 3 0.010 0.010 0.039 0.19 0.22 0.19
0.005
0.006
e
3
0.206
0.241
0.223
0,006
0.007
Sum represents equivalent molar concentration of n-hexane. Two moles o f propane, 1 mole of hexene, or 1 mole of benzene a r e each equivalent t o 1 mole of n-hexane. a
CALCULATIOK AND RESULTS
To determine sample composition the amount of hydrogen pumped away must be known as ne11 as composition and volume of the hydrocarbon concentrate. Volume of concent,rate is drtermined by the manometer, Jfs, and the known fixed volume of that section having the palladium tube. In expansion measurements this volume was found to be 50 ml. with the nieasurement made done with the palladium at 500" F. Since 125 ml. oi sample a t atmospheric pressure arc used, the quantity of lost hydrogen can be obtained by a pressure-volume calculation. The hydrocarbon concentrat,e is calculated in a rout.ine manner on an air-free basis. Nitrogen is corrected for the measured oxygen lost during its determination by absorption in pyrogallol. The most important question concerning the validity of this iuethod was whether or not representative samples could be mllected for mass spectrometric analysis. Specifically, separation by molecular weight might occur in the palladium tube nection and to study this possibility numerous synthetic blends of hydrogen, methane, and n-hutane were analyzed. S o separation was oherved. Results of one blend are shown in Table I. I n this case 0.011% each of methane and n-butane was determined within 0.005%. This was the only experiment carried out in this concentration range and such results could probably be improved. I n this (:me, 4 litcrs of sample were used to ohtain a hydrocarbon concent,rate. Reproducibilit,?. is illustrated in Tahle 11, where mean values for eight analyses of the same gas are presented. At concentrations of 0.064 to 0.45% standard deviations of 0.016 to 0.037y0 occur. Table I11 includes the mean analysis of 14 synthetic
blends each made up separately to 98.81% hydrogen, 0.61 % methanr, and 0.57% n-butane. Thr standard error of 0.074% for methane and 0.102% for n-hut'ane includes possible errors in syuthesizing samples as well as analytical errors. iinalyses of samples containing Si.02% hydrogen, 1.455% methane, 0.035% ethane, and 1.49% n-butane are also t.abulated in this table. Here, a standard error of 0.082% occurred for methanr and 0.102% for n-butane. Samples containing hydrogen, methitne, and n-butane indicated the ability of the method to determine hydrocarbons over t,he C, t,o Ci. range wit,hout loss due to handling. secondary problem was discowred during a study of synthet,ic tdends containing small amounts of n-hexane. Results of suc*ha st.udy are shown in Table IV. Itcfcrerice to these data shows that in addition to n-hexanr, hcreires and/or carbon-6 cyclops,r;tffins, henzene and prop:iiie were determined. These ext.raneous hytlrocarbons w e apparently the result, of thermal decomposition ( 2 ) and rcgreaciit about 10% of the n-hexane determined. These data bverc ohtained with the palladium t,ube operated a t 600" F. 111 a11 attempt to reduce this effect the operating temperature WLLF l o a n e d t.o 500" F. and Teflon t~ape \vas wrapped around the Kovar and stainless steel ring of the palladium tube assembly (Figure 1j. The thermal decomposition for this arrangement appeared t.o reniain about relative lo%, however. Palladium by itself a t 500" F. was found to produce 5 % decomposition, so that apparently no complete solution exists €or this problem. However, a 10% decomposition of Cg and C, components causes an insignificant error due to the low level of absolute concentrations encountered in samples. Furthermore, the decomposition products themselves are determined. No actual weight, loss of total hydrocarbons occurs other than that due to the small amount of hydrogen formed during deconiposition. LITER 4 I L l R E CITED
Brown, 11. A , , and Skahan. D. . I , A N ~ LCHEM., . 26, 788 (1954). Coquillon, J. J., Compt. rend., 77, 414 (1873). Fleiger, A. G., IND. ESG. CHEM.,A N ~ LED., . 10, 544 (1838). Lombard, V., and Eichner, C., Coinpt. rend., 195, 322 (1932). Marshall, AI. J., and Constabaris. G., Can. J . Chem., 31, 843 (1953). Taylor, R. C., and Young, W.S.,ISD. ENG.CHEM.,ANAL.ED., 17, 811 (1945). R E C E I V E Dfor review August 24, 1R54. Accepted November 17, 1954.
Colloidal Glass Suspensions for Use as Standards for Measurement of Thymol Turbidity JOHN G. REINHOLD The W i l l i a m Pepper Laboratory o f Clinical Medicine, Hospital o f the University o f Pennsy/vani+, Philadelphia 4, Pa.
The suitability of colloidal glass suspensions for standardization of thymol turbidity measurements made by tneans of photoelectric spectrophotometers has been investigated. The preparation of a semipermanent standard resembling in its behavior the suspension formed in the thymol test together with its application to the calibration of photoelectric photometers for use in the thymol turbidity test is described. The readings of the turbid suspension produced by the reaction of the thymol-barbital reagent with blood serum vary in photoelectric photometers and spectrophotometers of different design. These variations are related in part to the composition of the serum.
T
HE lack of a satisfactory standard for measurement of thymol turbidity (8) in photoelectric photometers has caused considerable confusion. The use of barium sulfate suspensions for this purpose was proposed by Shank and Hoagland ( 1 4 ) . Such suspensions are unstable and their reproducibility has been uncertain. A more serious difficulty arising in connection with the use of such standards was the erroneous description by Shank and Hoagland of the concentration of barium chloride used for preparation of the barium sulfate standard. This was said to be 0.0962Ar, rvhereas actually the corresponding molar concentration had been used. Thus, at the time t h a t mea~urenientof thymol turbidity was adapted to photoelectric photometry two standards came into
ANALYTICAL CHEMISTRY
240 use. T h a t consisting of barium sulfate prepared from 0.0962M barium chloride conformed to the readings obtained by use of the gelatin-formazine-egg albumin standards originally recommended by Maclagan (8). T h e second, prepared from 0.0962.V barium chloride, gave thymol turbidity readings twice as high. T h e error was corrected, subsequently, but many who used this method remained unaware of the correction. Actually the majority in this country appear to be using standards based on the smaller units. For convenience, these have been called ShankHoagland units, to distinguish them from the units obtained by Maclagan's calculation. Copper sulfate ( 3 ) and Evans blue ( 7 ) also have been used to some extent as standards. Both have the disadvantage of giving readings that depend directly upon the width of the spectral band as well as other optical characteristics of the photometer. These vary widely in the photometers ordinarily used in clinical laboratories. Readings of turbid suspensions are not nearly as dependent upon wave length. hloreover, the light intensity and the arrangement of the optical system have an important influence on turbidity readings. A suitable turbid suspension simulates more nearly the conditions affecting measurements of suspensions produced in a turbidity test than does a colored solution. Glass suspensions have been in use for some time for serological and bacteriological studies as a means for estimating turbidities, and it appeared likely t h a t they could be used for standardizing thymol turbidity measurements. Brewer and Cook (1)and later Hallinan ( 4 ) have described the preparation of bacteriological standards. Unfortunately, such colloidal suspensions vary widely in particle size and in their optical properties. T h e method proposed by Hallinan ( 4 ) for their standardization consisted of the preparation of successive dilutions of a colloidal suspension, from which larger particles had been removed by filtration through paper, to give an arbitrarily selected reading in a Klett-Summerson photoelectric colorimeter. Trial of this method proved it to be inadequate for standardization of thymol turbidity and other quantitative turbidity measurements currently used in clinical chemistry. The absorbances of such suspensions measured in various widely used spectrophotometers differed extensively and unpredictably and showed no consistent relationship to thymol turbidity. Studies have been made of colloidal glass suspensions with the object of devising a method for obtaining a preparation suitable for use as a turbidity standard, particularly for thymol turbidity measurements. As a result of these studies, a colloidal glass suspension has been obtained t h a t resembles in its behavior the turbid suspensions produced by reaction of serum with the thymolbarbital or certain other reagents. Its equivalence in terms of thymol turbidity has been established by several methods, and its reproducibility and other characteriqtics of its behavior have been investigated. PROCEDURES
Thymol turbidity was measured 30 minutes after addition of 0.1 ml. of serum t o 6 ml. of thymol-barbital reagent. I n the earlier part of the study the reagent \$as prepared according to Maclagan (8); later a modified technique developed in this laboratory was substituted ( I S ) . T h e reagent was brought to 25" C. before addition of serum and was maintained within 0.5" of this temperature in a constant temperature during the 30minute incubation period (15). Readings were made a t room temperature which varied from 23" to 28" C. T h e thymolbarbital reagent was used for adjusting the photometer to zero. Photometric readings were made by means of two Beckman Model D U spectrophotometers; two Evelyn photocolorimeters equipped with standard reflectors; a third Evelyn instrument with a dull mat reflector; and a fourth with a polished reflector a s supplied by the makers for use with solutions of low transmittance; three Klett-Summerson photocolorimeters; and two Coleman, Jr., spectrophotometers. A few readings have been made using a Bausch and Lomb photoelectric spectrophotometer.
/
*Be chrnan 0.407
T
0.30
I
0 2 0 -i 5T
3
0 19T a 20T 0 22T 0 t 3 T
3PREPS.
INIH
z3c CENTRIFU. a MIN.
23c.
23C.
5 I'
10
" 'I
A HOMOGENEOUS
DOTTOM FRACTION TOP E MIDDLE
A i
0.10
I 0.20
-*EvaIzjn Figure 1.
I 0.30
-
I040
(bright r e f l e c t o r ) .4bsorbances of colloidal glass suspensions
B e c k m a n spectrophotometer measurements compared w i t h Evelj n photocolorimeter equipped w i t h polished reflector
Table I. Daysa
11
6
9 0 52 0
a Age
Effect of Preservatives on Stability of Colloidal Glass Suspensions Preservative Sodium benzoate, 0 1 % Rlerthiolate, 0 01% Sodium borate, 1%
No Dreserl-ative
Absorbance 0.072 0,060 0.131 0.086 0.193 0.136 0 193
of dilution of colloidal glass tested
T h e Beckman spectrophotometer 1 eadings were made using 10-mm. cuvettes and the red-sensitive phototube. Slit width settings ranging from 0.04 to 0.30 had no effect on the measurements. Cuvettes of 20-mm. diameter n ere used in the Coleman, Jr., spectrophotometer, and in the Bausch and Lomb instrument these measured 18 mm. All instruments were adjusted to a zero reading with distilled water when glass suspensions n ere studied. Thymol turbidity measurements were made after the instrument had been adjusted to zero by means of a cuvette containing the reagent. All measurements mere made a t 660 nip. Results of photometric measurements are enpressed either a s absorbances or as Shank-Hoagland units of turbidity as defined above. T h e use of Shank-Hoagland units in preference to Alaclagan units has been recommended by the Commission on Liver Disease of the Armed Forces Epidemiological Board as one step toward accomplishing a uniform system of reporting results of turbidity tests used in clinical laboratories. EXPERIMEYTA L
Preparation of Stock Colloidal Glass Suspension. A borosilicate glass reagent bottle of 1-liter capacity fitted with a glass stopper is filled t o about one fourth its capacity with fragments of clean borosilicate glass. After being rinsed several times with distilled water, the glass fragments are covered with distilled water and agitated vigorously on a mechanical shaker until a milky suspension is produced. The time varies with the type of shaker, rate, and force of oscillation; 8 to 12 hours have been sufficient when a reciprocal shaker making an excursion of 8 inches 150 times a minute mas used. The suspended glass is decanted into a cylinder of 1000-ml. capacity and diluted to the mark with distilled water. It is mixed and allowed to stand 7 2
V O L U M E 27, NO. 2, F E B R U A R Y 1 9 5 5 hours. The uppermost 300 ml., which includes the more finely divided material, is decanted. As a n alternative method, the top 300 ml. may be divided among six conical centrifuge tubes of 50-ml. capacity. These are centrifuged a t 1100 r.p.m. for 8 minutes in a centrifuge having a radius of 50 em. The suspension i p decanted into a clean borosilicate bottle fitted with a glass stopper. Properties of Colloidal Glass Suspensions. STABILITY. The particles of glass settle very slowly, little sediment being visible on the bottom of the container after 24 hours. S o change occurs during the time required for measurements of the ahsorbance. PERMAYENCE. The permanence of colloidal glass standards has been studied over periods ranging u p t o 18months. I n some preparations a decrease in turbidity, equivalent to 0.5 to 1% per month occurred during the first months. Others remained unchanged indefinitely. The gi owth of microorganisms can cause a rapid fall in absorbance. Addition of preservatives, with one exception, has accelerated deterioration (Table I), as has the presence of other solutes (Table 11). The alkaline reaction of the suspensions of higher absorbance (0.30 upwards) inhibits growth and probably aids in stabilizing the suspensions. The use of a solution of mercuric chloride containing 0.1 gram per liter appears to have'no deleterious effect on stability of dilute suspensions and yet checks growth. Information concerning stability of mercuric chloride-preserved standards over long periods is not yet available. Until such information is available, the use of freshly distilled water for preparing dilutions is recommended. Containers must be chemically clean. T h e p H affects stability (Table 11). Suspensions buffered at approximately neutral p H deteriorated slo\vly. At pH values ranging from 4.0 to 6.0, deterioration was rapid. Fractionation of Colloidal Glass Suspensions and Selection of a Suitable Preparation. The method of preparation described by Hallinan ( 4 ) produces suspensions with a \vide range of particle sizes, Such preparations when measured in different photometers or under different conditions of illun~ination varied considerably in their readings. Furthermore, their behavior differed from that of the suspensions produced by serum in the
*//
0,400
0,350 0,300
0.250 0.200 0.150
0.100
I CLEAR, NO FLCCCULA1IOI.I 0 LACTESCENCE ; A N Y DEGREE
0.050
@ LPICTESCENCE G FLOCCULATION COLLCID&L G L R 5 5 PREP 231
FLOCCULATION
-1-1-1-1-I-I5
10
15
Thymol Turbidity
Figure 2. Influence of composition of serum upon absorbances of thymol turbidity tests Clear and lactescent sera and sera that subsequently showed flocculation
241
Tahle 11. Effect of pHBnd l'arious Buffers on Stability of Colloidal Glass Suspensions Daysa 4 11 0 10 15 90 0 10 15
90 0 3 0 3 0 3 0
pH 6.9
Distilled water exposed to air 4 weeks
6.9
Freshly distilled water, low in COS
7.3
Phthalate, 0.05-11
4.0
Acetate, 0 1-11
5,O
Phosphate, 0.066.11
6.0
Phosphate, 0 066.11
6.9
90 0
Phosphate, 0.06G.11
7.4
16 90 0
Sodium borate, 0.05.11
9.2
10 15 in ~.
3 a
Buffer Sone
Absorbance 0.139 0.136 0.112 0.114 0.108 0.109 0.112 0.115 0 112 0.111 0.122 0.082 0.131 0.103
0.135 0.117 0,109 0,105 0.102 0.088 0.109 0.102
0,094 0.086 0.130
0.139
Age of dilution of colloidal g h s tested
thymol test. It was not possible therefore t o obtain satisfactory 5tandards until preparations having a particle size range rcsemtiling the suspensions formed by the ieaction of thymol-barbital x i t h serum protein and lipides n el e isolated. I n order to accomplish this, glass suspensions lvere alloucd to sediment in glass cylinders of 1-liter capacity. After 2 t o 4 meeke, a qeries of zones, six to eight in number, each consisting of a fairly homogeneous range of particle sizes and varying in volume from 75 to 100 ml., could be didinguished. These zones w r c lemoved by aspiration and examined separately in several photorneters. Their absorbances nere compared with those of the turbid suspensions formed in the thymol turbidity test. Such evaluations were aided by the finding that the ratio of absorbance measured in the Evelyn photometer a t 660 mfi t o that mrasuied in the Beckman Model DU specti ophotometer \$as determined by particle size. .%]though numerous trials n ere made of individual zones 1emoved at various levels from different preparations of colloidal glaqs, none of t h e sepaiated zones resembled in its optiral behavior t h a t of the thyniol-serum ieaction product. This suggepted that a variety of particles of differing sizes werc being formed in the latter. Proceeding on this assumption, conibinations of two and three separate zones rvere tested nmt. .I Satisfactory identity with the thymol suspensions waR 6rially achieved by separating the uppei 300 nil. of a glass suspension that had been allowed t o sediment under the influence of gravity for approximately 7 2 hours. A similar preparation could be .obtained much more rapidly by an alternative method, centrifugation a t 1100 r.p.m. for 8 minutes as described above. However, the gravity method has provided more uniform preparations (Figure 1). Figure 1 shows the relationship between the absorbances of a Ceries of colloidal glass suspensions as measured on the Beckman spectrophotometer and an Evelyn photocolorimeter, the latter equipped with a polished reflector. The solid line shows thc regression of gravity separated top fractions. The regression of thc readings of thymol turbidities of specimens that subsequently showed flocculation is shown by the upper line; that of lactescent specimens, excepting those that flocculated, is shown by the lower line (Figure 2). Both the T (gravity separated) and C (centrifuged) series of glass suspensions fall between the t n o lines. Thirteen dilutions derived from eight different preparations are included. The greatest deviation in this group is about 5%, evidence that preparations can be I eproduced successfully. Several preparations derived by methods other than that J-icld-
ANALYTICAL CHEMISTRY
242 ing the T series are also shown (by open arid solid triangles) in Figrtre 1 . These include the middle ahd bottom cuts of preparation 23. Both depart from the relationship characteri8tic of the suspension (shown by top fraction. A sample of a colloidal gl the semicircle) prepared by the Sational Institutes of Health foi. standardization of vaccines arid related applications, also de~ thymol turbidity readings. partled from the limits defined l ) the Heterogeneous Nature of Thymol Reaction and Significance for Measurement of Turbidity. The first attempts made in this study to establish a relat’ionship between colloidal glass suspensions and thymol turbidity yielded disconcertingly variable results, especially when the thymol turbidity was measured by use of an Evelyn photocolorimetrr equipped with a bright reflertor. Ratios of thymol turbidity to Beckman readings ranged froin 65 to 18 (Figures 2 and 5). Siini1:ir relationships xere seen \vheii certain other photometers were used. The possibilit,y that thew differences in optical behavior w-ere related to reaction of th(3 several serum constituents knon-ii to contribute to the turbidit!, was investigated. I t ip wEll established that in addition t o gamma globulin [Maclagan and Bunn (IO)]and 8-globulin [IU spectrophotometer.
flocculation gave ratios that fell between those of the flocculent and lactescent specimens, although much closer to the formei. 3s shown by the solid line in Figure 2. Table 111shows the equxtions for the regression of the three groups of measurements calculated by the method of least squares. The regression coefficients of the lactescent and flocculent groups tested statistically differed significantly. Readings made with the Beckman spectrophotometer when compared M ith those of Evelyn photometers (equipped with qtandard ieflectors) and by means of Coleman, Jr., spectrophotometers showed a similar spread, again depending upon the composition of the serum (Figures 3 and 4 ) . In Figure 3, the regression of flocculent sera fell along the upper line, as did that of lactescent sera that gave rise to flocculation. The regression of sera either clear or lactescent that did not produce flocculation fell along the lolyer broken line. ri further decrease in light intensity in the Evelyn instrument brought about by the w e of n dull mat reflector had little effect on the readings. The colloidal glass prepai ation selected for use as standard was one that when tested by this method gave readings that appeared on the graph midway between the regression lines of the flocculating and lactescent sera as shown by the broken line with the two dots between dashes in Figures 3 and 4. The solid square shows readings of the National Institutes of Health’s colloidal glass suspension, and the partially shaded squares those of preparation 23T. The difference between the thymol turbidity readings of the two tvpes of sera is frequently sufficiently large and consistent to enable the thymol turbidities predominantly caused by change in protein (the flocculation type), to be distinguished from those apparently due to elevation of lipide (the lactescent type). I t i* probable that thesr may differ in clinical significance as well as chemical. Figure 5 shows the frequencies with Mhich ratios of thymol turbidity to absorbance occhred in “flocculating” as
,
0.30
A E w l y n - S t a n d a r d reflector
Figure 3. Distribution of thymol turbidit! readings Readings with Eveltn photometer equipped with btundard reflector plotted against Beckman spectrophotometer readings. Symbols have same meaning as in Figure 2. Oval. represent thymol turbiditir. done in buffer of pll i . 8 0
I
0
/
’
I n order to test the effect of a predominance of a reaction xith the protein components in certain sera as compared with predominance of lipide reaction in others, thymol turbidities of sera that subsequently showed flocculation on standing with the thymol-barbit,al reagent were measured in both photometers. The ratios of absorbances p1ott)ed as sh0n.n in Figure 2 fell along the upper broken line. The thymol turbidity readings of lactescent sera, known to be high in lipide, were plotted in the same way. The ratios of the latter fell along a line of considerable less slope, the lower line in Figure 2 . Some, but not much overlapping of the flocculation group occurred. When lactescence was associated with flocculation, the latter determined the location of the point of the graph. Clear sera that did not show
0.10
Figure 4.
oko
O.$O
AcolernanInfluence of serum composition on absorbance
Same relationships as in Figures 2 and 3, except that Coleman, Jr., spectrophotometer used to measure absorbances
V O L U M E 2 7 , N O . 2, F E B R U A R Y 1 9 5 5 Bright reflector
i i Lact I
3
243 pensions prepared by the technique described above and their equivalence in units of turbidity can be derived from Figures 2, 6, and 7 and Table IV. The thymol turbidity readings used for these calibrations were obtained by means of an Evelyn photocolorimeter equipped with a polished reflector. This instrument JT-as calibrated by equating the rtsults of some 500 readings made by the method originally proposed by hIaclagan ( 8 ) ,the Kingsbury, Clark, Williams, and Post turbidity standards for estimation of urine protein, with the absorbances obtained by use of the EvelJn instrument. The equivalent of the urine protein reading obtained from the Kingsbury standards was divided by 60 to obtain thymol turbidity in Maclagan units. This calibration was verified by averaging the readings of 25 different preparations of barium sulfate prepared according to the directions of Shank and Hoagland ( 1 4 ) ;0.0962M barium chloride TTas used. The results of the two methods of calibration, doubled in order to obtain Shank-Hoagland units, were identical. The resulting calibration gave values 5% lower than those derived by calculation of the mode of the measurements of a suspension of colloidal glass made by 45 different laboratories (12). This is not a significant difference.
Standard r e f l e c t o r lo;
V
A
Figure 5. Frequency of occurrence of given ratios of thymol turbidities to absorbances i n different t? pes of sera
030
I
3
.
0 2 j T
compared with lactescent sera. The former group is homogeneous. but in the “lactescent” and “clear” groups that failed to show flocculation the distribution is bimodal. This finding suggests that low ratios should be regarded as a n indication of ahnorin:ility of the type associat,ed n-ith liver disease. Present methods of cvaluating the t,hymol test fail to demonstrate such abnormality in many specimens. Further studies of the clinical potentialitic+ of this observation are planned. Standardization of Colloidal Glass Susp-nsions. The ahsoil)a n w of the stork glass suspension is measured by using a wave length of 660 mp. K a t e r is used for adjusting the photometer to zero. If the absorbance exceeds 0.30, it is diluted with freshly dietilled ivater sufficiently to give an absorbance of approximately 0.1.5. The cuvette should be similar in its optical charactei.istirs to those used for the thymol or other tests. The qualit,! of the di%tilled water used for diluting the concent’rated suspensions may he important. Freshly distilled Tvater is preferred to tieionizer1 water. Many different types of photoclcctric photometers are currently used i n clinical chemistry, and one of the necessities for a suitahle standard is t,hat it give similar results in several. a t least, of the more ividely used instruments. Test? made of a group of photometers intiicnteti t,hat absorbance nieus~u‘ements agreed in some but differed significant,ly in others. The differences betlveen Beelininn DU spectrophotometer readings and thoEe of Evelyn and C’olrnian, Jr., phot,ometers have been described; such differences wcre relat,ed in part to the vomposition of the serum and type of reaction. The relationship between absorbance of the colloidal glass sus-
Table IV. Average Ratios of Thymol Turbidit) to .ibsorbance with Several Spectrophotometers
Beckman, DC Coleman, J r . Evelyn, standard reflector Evelyn, polished reflector Klett-Summerson
KO of Specimens 09 66 C;u
62 41
TT/A Mean 45.2 44.1 42.4 58.7 0.137
-
Range 27-60 32-54 34-49 52-61 0.10-0 22
I
e
,
a*
3
I I.’ I-_
L-
__- 1 - 1
50
I“. 3
I-
15.0
TT
Figure 6. Relationship between thymol turbidity and absorbance with the Coleman, J r . , spectrophotometcr S, mbuls have
came meaning az in Figure 2
Figure 6 shows meawrements made by use of Coleman, Jr., spectrophotometer Absorbances are plotted against thymol turbidity. The readings of several glass suspensions are included These readings were nearly identical n ith those found by use of the Evelyn instrument. Table Is’ shows the averaged values of the ratio of thvmol turbidity to absorbance of 41 to 69 specimens of serum together v, ith the range of variation meawred by instruments of different design The factors in Table IV were obtained in each instance by u-c of severil instruments of a given make, and the ratios of thymol turbidity and absorbance, therefore, may be regarded as reprewntative. Thymol turhidity may be calculated by multiplliny the absorbance by the ratio of thymol turbidity to absorbance. However, it is advisable to calibrate each instrument by means of a standard colloidal glass suspension rather than to depend on factors derived in other laboratories. When a glass suspension is used as a standard, thymol turbidity T, is calculated as follows: T , = A , -where T , represents the units
A*
of turbidity of the glass suspension, A , its absorbance at 660 mr, and T,/A, the mean ratio of thymol turbidities to absorbances tabulated in Table Is’.
244
ANALYTICAL CHEMISTRY
T o obtain the thymol turbidity, T?', of a serum specimen, the following calculation is used: m
where -4is the absorbance of the turbid suspension produced by reaction of serum with thymol-barbital reagent and T , and A , have the same significance as in the preceding equation. The regression equations calculated for the values in Figuies 1 to 4 and 6 and i indicate that the origins are sufficiently close to zero to allow intercepts to be disregarded. Thymol-barbital reagent is used to establish the zero setting when thymol turbidity is measured, but water is used for this setting when the glass suspensions are read. The influence of changing the optical system in the Evelyn photometer is shown in Figure 7. Thymol turbidities expressed as absorbances meawred in an Evelyn photometer equipped n ith a polished reflector are plotted against absorbances measured in a similar instrument n i t h an unpolished reflector of the type supplied as standard equipment. The absorbances obtained by use of the bright reflector are approximately three fourths those of the standard instrument. However, the colloidal glass suspension mas affected nearly to the same extent as were the thymol turbidities, so that measurements referred to the glass standard would have differed by no more than 10%. There is little difference between the behavior of lactescent sera as compared with flocculating sera produced by this alteration in the C w l y n instrument DISCUSSION
The results of photometric measurements of thymol turbidity will vary, depending upon the light intensity and the arrangement of the optical system of the photometer used. The extent of the effect upon the readings varies in different instruments. Thus, the Evelyn photoelectric colorimeter and the Coleman, Jr., spectrophotometer gave similar results for thymol turbidity readings. The Nett-Sammerson photoelectric colorimeter and Beckman Model DE spectrophotometer gave results that often differed from those of the other two instruments. These differences, particularly of the Beckman instrument readings, depended upon the predominance of the reaction of protein a? opposed to lipide components of serum with the thymol-barbital reagent. The importance of the instrument used for photometric readings of thymol turbidity has been pointed out by Hoyer and Jorgensen (6). However, the shortcomings of visual measurements of thymol turbidity are far greater than those of photometric (12). The values derived from readings of standards used for calibration of the photometer or for direct comparison in visual measurements also are affected by the instrument or, in the case of visual comparison, by the light intensity and the positions of light, samples, and observer. This explains, in part, the large differences found between the results of different laboratories ( 1 2 ) despite the use of a standard such as barium sulfate. However, the well-knor\ n difficulties of reproducing suspensions of this substance are undoubtedly the most important cause of such variations. Maclagan (9) has replaced the egg albumin-formazine-gelatin standards originally proposed with a standard prepared by treatment of diluted serum of known protein content with sulfosalicylic acid. The author lacks sufficient experience with this standard to enable evaluation of its reproducibility or conformity of the particles produced to the size and surface area of those produced in the thymol test. Variations arising from differences in compositions of the serum and technique of combining serum and reagent are to be expected. Such a standard also lacks permanence. The suspension of colloidal glass described in this paper offers
0
AEvelyn (std.rehector)
-
0.30
0.20
-A ~ v O.'lO e l y n( b r i g h t reflector) 0.30
Figure 7. Absorbances of thymol turbidities Measurements w i t h Eveltn photometer equipped w i t h standard reflector compared w i t h those f o u n d hy similar i n s t r u m e n t w i t h standard reflector
the advantages of semipermanence and of resembling more nearly than other standards in general use the particle size and optical behavior of the turbid suspension produced in the thymol test. The suspensions of colloidal glass diluted with 0 01 yo mercuric chloride solution have remained unchanged for one year. The use of this solution rather than distilled water therefore is rpcommended for dilution of the stork suspension of colloidal glaFe. ACKSOW LEDGXIENT
The author is indebted to Charles Jones, Veterans Hospital, S e w Orleans, La., for the suggestion that colloidal glass suspensions might be utilized for standardization of thymol turbidity readings, and to Roderick Murray, Divieion of Biologics Control, Sational Institutes of Health, Bethesda, Md., for a sample of a colloidal glass suspension prepared by the National Institutes of Health. LITER4TURE CITED
Brewer, J. IT., and Cook, E. H. AI., ,4m.J . Pub. Health, 29, 1147 (1939).
Cohen, P. P., and Thoinpson, F. L., J . Lab. Clin. M e d . , 32, 475 (1047).
Ducci, H., I b i d . , p. 1266. Hallinan, E'. J., -4m. J . Pub. Hcalth, 33, 137 (1943). Hoyer. G., and Jorgensen, J., Scand. J . C2in. & Lab. I I I L ' C B 4,~ . , 319 (1952).
Kunkel, H. G., and Hoagland, C. L., J . Clin. Inrest., 26, 1060 (1947).
Kunkel,' H. G., and Hoagland, C. L., Proc. SOC.Ezptl. Biol. Med., 62, 2% (1946). Naclagan, S . F., Brit.J . Erpll. Pathol., 25, 234 (1944). Alaclagan, 37. F., Brit. M c d . J . . 2 , 197 (1947). Naclagan, X. F., and Bunn, D.. Biochem. J . (London),41, 580 (1947).
Recant, L., Chargaff, E., and Hanger, F. AI., Proc. SOC.E.rpt2. Bzol. Med., 60, 245 (1045).
Reinhold, J. G., A m . J . C1iu.Pathol., to be submitted. Reinhold, J. G., and Yonan. V. L.. Clin. Chemist, 5, 82 (1953). Shank, R. E., and Hoagland, C. L . J . Bzol. Chem., 162, 133 (1946). Yonan, V. L., and Reinhold, J. G , Am. J . Clin. Path02 , 24, 232 (1954). R E C E I V Efor D review July 15, 1954. Accepted October 25, 1954. Presented in part before the Division of Biological Chemistry at the 121st Meeting of the AMERICAN CHEMICAL SOCIETY, Jlilwaukee, Wis., April 1952. Work sponsored b y the Commission on Liver Disease of the Armed Forces Epidemiological Board and supported by the O f f i c ~of the Surgeon General, Department of the .4rmy.
