Determination and Characterization of Gallotannin by High Performance Liquid Chromatography Thomas H. Beasley, Sr., Howard W. Zlegler," and Alexis D. Bell Corporate Analytical Services Laboratory, Mallinckrodt, Inc., P.O. Box 5439, St. Louis, Mo. 63 147
-. Gallotannln In tannlc acid, and In source materials, Is determined by high performance llquid chromatography (HPLC). Characterization is achieved by the chromatographlc separatlon and quantlficatlon of the various galloyl glucoses found In tannln. Sample solution, plus hydroquinone as an Internal standard, Is Introducedonto a Corasll II column whlch Is gradient eluted with a solution of tetrahydrofuran-methanolglaclal acetlc acid In hexane. The absorbances are continuously monitored at 280 nm, uslng a flow-through ultraviolet double-beam photometer. Pentagalloyl glucose, prepared by methanolysis of Oriental gallotannln, Is used as a reference sample. The relatlve standard devlatlon was found to be 2.5 % absolute. Orlental and sumac tannln were found to be essentlally Identlcal. Aleppo tannln dlsplayed dlfferlng characterlstlca wlth a lower molecular welght.
Gallotannins are classed as the hydrolyzable vegetable tannins which yield gallic acid as a hydrolysis product (1).The major commercial sources of gallotannins are: a) Chinese or Oriental gall nuts which form on the leaves of Rhus semialata; b) Aleppo nuts, from Turkey and North Africa, found on the wood of Quercus infectoria;and c) sumac leaves of Rhus coriaria and Rhus typhina, grown chiefly in Sicily and the Mediterranean area. The gallotannins are very important commercial products. The technical, water-extracted grades are used primarily for tanning leather. The solvent extracted, purified grades have wide reagent, medicinal, food, and beverage usage. The various sources of gallotannin (or tannin) yield products with differing chemical structures. Based on a review of the literature, Haslam (2) reports that Oriental and sumac tannins are polygalloyl glucose derivatives with a ratio of glucose to gallic acid of 1 to 9 or 10. Turkish gall nuts yield a tannin with a ratio of 1 glucose to 5 or 6 gallic acids. Fischer ( 3 ) ,in the early 19OO's, synthesized depsides and tannins. He concluded that Chinese and Turkish tannins were structurally different. He stated that pentadigalloyl glucose is closely related to tannin in all its properties. Haslam and co-workers ( 4 ) corroborated Fischer's conclusion based on methanolysis at pH 5-6. Several inconsistent theories have developed regarding the chemical composition of tannins. Recent chromatographic work, as reported by Armitage et al. ( 5 ) ,concludes that Oriental tannin is a mixture of penta-, hexa-, hepta-, octa-, and nonagalloyl glucose. Free gallic acids are also present in tannins in varying amounts, depending on the degree of hydrolysis. Tannin has been chromatographed by most of the known techniques. This paper reports the practical analytical separation, characterization, and quantification of tannins by high performance liquid chromatography (HPLC), using a propietary silica gel column packing, with gradient elution. The equipment is identical to that reported by us for the assay of opium alkaloids (61,except for the size of the column, the flow cell, and the wavelength used. Our chromatograph wa8 fully automated wing pneumatically actuated sequencing valves. All data were reduced ueing a dedicated minicomputer. 238
0
Tannin hydrolyzes readily in aqueous solution. We found tannin to be unstable in organic solvents containing more than about 1%water. To circumvent hydrolysis during extraction andlor dissolution, we focused our attention on anhydrous solvents. Acetone, methanol, ethyl acetate, ethanol, and acetonitrile all showed various artifact peaks even when anhydrous. Distilled tetrahydrofuran (dry) met all the criteria necessary for our experiments. The addition of 1%hydroquinone serves the dual function of an internal standard and an antioxidant (to prevent explosive peroxide formation). Tannin from Turkish gall nuts is stable in the HPLC program solvent for several weeks while Oriental and sumac tannins form artifacts in a few days.
EXPERIMENTAL Apparatus. The HPLC instrument used was a hybrid consisting of: a) a double-beam ultraviolet detector (280 nm) with a 2-mm path length flowcell (Altex Model 161); b) a modified Milton Roy miniPump and dampener (from Glenco Scientific, Houston); c) a 10-mV full scale recorder; d) a 2 X 500-mm microbore glass column, filled by hand tapping, with Corasil I1 (obtained from Waters Associates);e) a stream sampling valve (Altex);f) a 5-pl sample injection valve; g) and 2- and 3-way valves, tees, crosses, and connecting tubing required for assembly (all obtained from Laboratory Data Control, Riviera Beach, Fla). A closed bulb gradient elution system, connected to the stream sampling valve, was an integral part of the chromatograph. A complete listing of the component parts, a schematic diagram, instrument operating parameters, and construction of the gradient elution cell are described in our publication on the assay of opium alkaloids using HPLC (6).A Hewlett-Packard Model 3352BLab Data System equipped with Lab Basic Software was used for data reduction and the computations. Reagents and Solutions. Reagent grade glacial acetic acid, gallic acid, and tetrahydrofuran (freshly distilled); ChromAR or SpectrAR grade hexanes and methyl alcohol; and purified hydroquinone were used. All reagents were from Mallinckrodt. Standards. Tannins extracted from Oriental and from Turkish gall nuts were purified in our laboratories to yield tannin standards containing less than 1%gallic acids (mono-, di-, and tri-). Pentagalloyl glucose (stripped tannin) was prepared from Oriental gall nuts according to the methanolysis procedure outlined by Haslam and coworkers ( 4 ) .Reagent grade gallic acid was used as a reference. All calibration standards were calculated on the anhydrous basis after actual determination of water by the Karl Fischer method as published by us (7). Program Solvent. Prepare by diluting 200 ml of distilled tetrahydrofuran to 800 ml with anhydrous methyl alcohol, and adding 8 ml of glacial acetic acid. Initial Mobile Phase Solution. Prepare by diluting 40 ml of program solvent to 1000 ml with hexane. Standard Solutions. 1)Mixed tannin standard solution: Dissolve 2.00 g of purified Oriental tannin, 2.00 g of purified Turkish tannin, and 1.00 g of hydroquinone (internal standard) in program solvent and dilute to 100 ml with program solvent. 2) Gallic acid standard solution: Dissolve 0.330 g of galllc acid reference material and 1.00 g of hydroquinone in program solvent and dilute to 100 ml with program solvent. 3) Pentagalloyl glucose standard solution: Transfer 0.500 g of pentagalloyl glucose to a 10-mlvolumetric flask, dissolve, and dilute to volume with gallic acid standard solution. All the standard solutions are usually stable for about one week. SamBle Preparation. 1)Gall nuts and sumac leaves: Transfer 4.0 g (for all gall nuts), or 6.0 g (for wmac leaves) of powdered sample to a 126-mlconical flask. Add 0.6 g of hydroquinone, accurately weighed, and 50 ml of program solvent. Stopper and agitate in an ultrasonic bath for 90 min. Filter through B glase Soxhlet thimble, fitted with
ANALYTICAL CHEMISTRY, VOL. 49, NO, 2, FEBRUARY 1077
a sintered glass frit (ASTM No. 40-60), using slight reduced pressure (the thimble may be tared to obtain a residue weight for material balance purposes). Calculations are made using the internal standard method. 2) Extracted (purified) tannins: Dissolve 4.0 g of sample in 100 ml of program solvent and agitate in an ultrasonic bath for 5-10 min. If results are to be calculated on the original basis, percent water by the Karl Fischer method must be determined. Results are calculated using area percents without reference to an internal standard. Procedure. Fill the gradient elution cell with initial mobile phase solution. Set the pump to a 2 ml/min flow rate. Load about 2 ml of prepared sample solution, or calibration standard solution, into the 5-gl automatic injection va!ve. Sample solutions extracted from gall nuts and sumac leaves must be filtered through glass fiber filters (MilliporeAP2501300 backup pads held in a Swinney-typefilter attached to a hypodermic syringe). Inject the sample solution into the chromatograph and commence the gradient elution cycle. Start the recorder set at a chart speed of 0.2 in./min. Run the chromatogram for 35 min. Empty and dry the gradient elution cell (with the stream sampling and other valves in the correct sequence position). Fill the cell again with initial mobile phase solution and the chromatograph will be ready for another sample or standard solution. The total cycle time, including re-equilibration, is approximately 50 min.
RESULTS AND DISCUSSION Typical chromatograms of tannin extracted from Oriental and from aleppo gall nuts are shown in Figure 1. Our first investigative problem was to attempt the qualitative identification or characterization of the many peaks in the chromatograms. The UV absorption spectra of each separated peak shows a maximum of 275 nm which is characteristic of gallic and/or tannic acids. Next we compared the retention times of as many available known substances, and their derivatives or decomposition products, which reportedly are constituents of natural source aleppo and Oriental tannin. Some of the products screened were: pyrogallic acid, gallic acid, digallic acid, sumac tannin, Chinese tannin, Korean tannin, aleppo tannin, tara tannin, methyl gallate, ellagic acid, chlorophylls, phloroglucinol, p-hydroxybenzoic acid, and quinic acid. The HPLC chromatograms of Chinese, Korean, and sumac tannin were essentially identical. Chromatograms of aleppo tannin were significantly different (Figure 1). Since these tannins fell into two basic types, we limited our experiments to them. In order to compare the two tannins, we prepared a 1:1 mixture by weight of Oriental and aleppo tannin with hydroquinone added as the reference. Figure 2A shows the chromatogram of this mixed tannin. No broadening of the peaks or any shoulders were observed, which is a strong indication that the two tannins differ primarily in the relative amounts of similar individual components. Each of the two types of tannin was subjected to methanolysis similar to that described by Armitage et al. ( 4 ) .Figure 3 shows the chromatograms of the two methanolysis products which we named stripped tannin because all the gallic acid is stripped from the molecule except that attached directly to the glucose moiety. Table I displays a summary of the results of the data and identities reported by Armitage et al. ( 5 )on stripped Chinese tannin and HPLC results on several of our stripped tannin preparations. While our HPLC data are not conclusive proof of peak identities, they are a very strong indication that the two major peaks in both stripped aleppo and stripped Oriental tannin are tetra- and pentagalloyl glucose, respectively. Subsequently, our stripped sumac tannin (see Figure 3A) was verified by an NMR procedure developed by Pearson and Spessard ( 8 ) , to be the P-penta-o-galloyl-Dglucose. Approximately the same retention time difference between each of the subsequent peaks was observed as between the tetra and penta peaks, which are known to differ by a single gallic acid unit. We named the subsequent peaks “hexa-, hepta-, octa-, nona-, and decagalloyl glucose”. Evaluation of the Oriental and aleppo tannin chromatograms
Figure 1. Typical HPLC chromatograms of tannin ( A ) Extracted from aleppo (Turkish)gall nuts, (B)extracted from
Oriental (Chi-
nese) gall nuts
I
w a
:: z
0
Lo
X N
a w
1 K
0
0
n C
i
IO
io
lk MINUTES
i5
io
$5
Figure 2. Reference chromatograms for identification purposes ( A ) Mixed aleppo and Oriental tannin
standard (internal standard is hydroquinone), (6) gallic acid standard, (C)pentagalloyl glucose standard (prepared from sumac tannin), (D)gradient programmed base line (Figure 1) indicates that Oriental tannin is mixed “heptathrough nona-”, and that aleppo tannin is mixed “penta- and hexagalloyl glucoses”, as stated by Haslam (2).Haslam ( 2 , 5 ) summarized the methanolysis studies of Turkish tannin and concluded that it was built on a mixture of the isomeric tetragalloyl glucoses in an unknown ratio. Our analysis of
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
239
FRESH ORIENTAL TANNIN SOLUTION
MINUTES
MINUTES
Flgure 3. Chromatograms of methanolysis product (stripped tannin)
Flgure 4. Artifact formation in Oriental tannin sample solution (methanol-THF-acetic acid)
(A) From sumac tannin, (6) from aleppo tannin
(A) Fresh solution, ( B )the same solution aged 2 weeks
Table I. Analysis of Methanolysis Products (Stripped Tannin) of Various Tannins Source
Tri-
Chinese gallsb Sumac leaves Korean galls Aleppo galls
.. .
... 1.8 15.3
Galloyl glucose, %a TetraPenta-
Hexa-
6.9 12.9 12.1
93.1 87.1 86.1
... ... ...
37.3
37.9
9.0
a Assayed by HPLC except as noted in footnote, b, below. Calculated from data presented by Armitage et al. ( 5 ) ,based on cellulose chromatographic separation and isolation with elemental analysis of fractions.
h
stripped Turkish tannin indicates that the Turkish tannin is built on a pair of galloyl glucose nuclei (see Figure 3 and Table I). Pentagalloyl glucose and the tetragalloyl glucoses are in a ratio of about 1:l.As Haslam ( 2 , 5 )reported, the tetra isomers (if both were present) were inseparable. No separation was observed by our HPLC. The triisomers are partially separated as indicated by widened peaks. Further, we noted that gallic, digallic, and trigallic acids in our system give approximate retention times directly proportional to the number of gallic acid units per molecule. A significant observation was that solutions of Oriental tannin in anhydrous acidified methanol-tetrahydrofuran usually form three artifacts (see Figure 4). We identified the first as methyl gallate, and the other two are probably methyl digallate and methyl trigallate, respectively. Aleppo tannin solutions under the same conditions form only a small methyl gallate peak, sometimes a very small second peak, but never a third. This is consistent with the pqstulated structures resulting from the fracture of the depside linkage by methanolysis. This indicates that a tridepside substituent is present to produce a methyl digallate leaving one gallic esterfied at 240
a glucose carbon. Very likely a tetradepside substituent is present to produce a methyl trigallate leaving one gallic esterfied at a glucose carbon. This indicates that the depside linkage nearest the glucose is slightly more labile than those farther out from the ester linkage. An experiment was devised to confirm our methyl gallate identifications and the prsposed direction of reaction. A sumac tannin methanolysis preparation was chromatographed after the first crystallization, before any purification (which would remove the methyl gallate). The chromatogram of this sample is shown in Figure 5. This trace shows a peak for each of the artifact peaks displayed in Figure 4.Therefore, we conclude that the first depside linkage out from the ester linkage is fractured preferentially. This gives methyl digallate from a tridepside substituent and methyl trigallate from a tetradepside substituent. Our constant volume, constant flow, gradient elution bulb does not produce a strictly linear solvent polarity program. Scott and Kucera (9) have defined the relationship mathematically as: % solvent
B in solvent A = (l-e-QT'v) X 100
(1)
where Q = flow rate in ml/min, T = time in min, and V = volume of the bulb in ml. The % solvent B (our program solvent) in solvent A (our initial mobile phase solution) was calculated for each peak retention time from the data in Figure 4B (See Table 11).A plot of the % program solvent in initial mobile phase for each peak retention vs. number of gallic acid units is shown in Figure 6. This plot is much more significant than one versus retention time which is logarithmic with respect to solvent polarity. The data points of Figure 6 show the following linear relationships between gallic acid units on molecules vs. gradient programmed mobile phase polarity: a) the methyl gallates; b) the gallic acids; c) a linear relation from tri- through hexagalloyl glucose. The aleppo tannin line also predicts two peaks
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
J
I J
i J
E
I
o t , . . . , . . : . , . . . . , . . . . , . . . . , . . . . , . . . . , . . . . ,, 0
5 10 15 20 25 30 35 40 GRADIENT-PERCENT PROGRAM SOLVENT IN INITIAL MOBILE PHASE
Figure 6. Linear relationships between gallic acid units on vs. gradient programmed mobile phase polarity
. . , , 45
molecules
( A ) Methyl gallates, (B)gallic acids, (C) aleppo tannin, (D) Oriental tannin, (0 highest molecular weights found in Oriental tannin Y
I
Figure 5. Chromatogram of methanolysis preparation before purification to remove methyl gallate reaction products
Table 11. Gradient Relationship vs. Retention Times Peak name Hydroquinone (int. std) Methyl gallate Gallic acid Methyl digallate Digallic acid Methyl trigallate Trigallic acid P-Gluco gallin Digalloyl glucose Trigalloyl glucose Tetragalloyl glucose Pentagalloyl glucose Hexagalloyl glucose Heptagalloyl glucose Octagalloyl glucose Nonagalloyl glucose Decagalloyl glucose Endecagalloyl glucose Dodecagalloyl glucose
Retention time, min
Gradient,” %BinA
4.1 4.8 7.0 9.0 10.3 11.8 13.0 14.2 15.2 16.5-16.8
7.9 9.2 13.1 16.5 18.6 21.0 22.9 24.7 26.2 28.3 30.4 32.8 34.8 36.6 38.2 39.6 41.0
18.1
19.9 21.4 22.8 24.1 25.2 26.4 27.5 28.5
42.3
43.4
B = Program solvent, A = initial mobile phase, flow rate = 2 ml/min.
a t one and two gallic acid units. Close inspection of the chromatogram in Figure 4B shows two very small peaks a t these positions. The literature lists two isomeric forms of trigalloyl glucose (2). The tri peak is a broad, barely resolved pair of peaks which a t low levels falls on either side of the average value on the line; d) a linear relation from hexa- to decagalloyl glucose; and, e) a linear relation from deca- through dodecagalloyl glucose.
Qualitative Identification. Separate reference chromatograms with hydroquinone as an internal standard, using the HPLC parameters above, of the following are prepared. 1) Mixed tannin standard solution. 2) Gallic acid standard solution. 3) Pentagalloyl glucose standard solution. Comparison of the three chromatograms, with sample runs, is made. The gallic acid and the pentagalloyl glucose peaks in the mixed standard solution chromatogram are identified (See Figure 2). The remaining peaks are readily identified using Figure 6 as a guide. The apparent column efficiency cannot be readily calculated for the tannin components because the separation was incomplete under our practical analytical conditions. For pentagalloyl glucose, prepared from sumac tannin, the peak is sufficiently defined to estimate the apparent column efficiency from Figure 2 as follows. Using classical calculations for calculating column efficiency, the height equivalent to theoretical plate was found to be 0.14 mm using 3490 plates in a 500-mm column. This would indicate the need of a higher efficiencymicroparticulate column. Tannin reacts very readily with metals, necessitating the use of glass and Teflon. We also found that the Corasil I1 column packing must be eluted with 12 N hydrochloric acid to remove traces of iron usually present before a column can be used. Before serious quantitative work was begun, the linearity of the absorbance monitor was checked. The absorbance a t 280 nm was found to be a linear function of the tannin concentration up to 0.32 absorbance unit, which corresponds to a tannin concentration of 5 pg/ml. For practical analytical work, a 0.5-mm cell for 100 kg/ml (of tannin) or a 2-mm cell for 25 kg/ml is recommended to obtain linear response. The average gallic acid response factor relative to hydroquinone was 0.440 using the recommended procedure. The tannin response for sumac tannin was 0.336 under these same conditions. Assuming that hepta-octa-nona is equivalent to octa, the gallic acid content of octagalloyl glucose is 87.1% by weight. The predicted tannin factor 0.440 X 0.871 (gallicfactor X gallic weight fraction) = 0.383 if the gallic acid units absorbance were strictly additive. The difference between the experimentally determined value and the estimate was 0.047, which is approximately 50 parts in 360 or 13% difference. In view of this, no major difference was expected between the tannin calibration factor for aleppo tannin and for sumac or Oriental tannin, and no statistically significant difference (2%
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
241
actual) was found between two such determinations (i.e., the aleppo factor RSD = 1.0%;sumac factor relative RSD = 1.7%). Therefore, the operating standard solution containing equal parts by weight of aleppo and Oriental tannin for both qualitative identification and quantitative calibration is justified. It is very inconvenient to maintain individual standard calibrations for each type of tannin. For semiquantitative work, a reasonable estimate for tannins is obtained by multiplying the gallic response factor by the weight fraction of gallic acid in the particular formula weight of tannin under consideration.
Table 111. Molecular Weights for Computation of Weighted Average Molecular Weight
Trigalloyl glucose Tetragalloyl glucose Pentagalloyl glucose Hexagalloyl glucose Heptagalloyl glucose Octagalloyl glucose Nonagalloyl glucose Decagalloyl glucose Endecagalloyl glucose Dodecagalloyl glucose
CALCULATIONS Calibrations. The gallic acid calibration must be determined first since pure tannins are difficult to prepare and maintain essentially free from gallic acids. Therefore, the small amount (usually about 1%)present in the mixed tannin standard must be determined and subtracted. Gallic Acid Calibration Curve. 1)From the chromatogram of the gallic acid standard solution, calculate the ratio of the gallic acid peak area to the peak area of the hydroquinone internal standard. 2) Calculate the weight ratio for the gallic acid by dividing the mg of anhydrous gallic acid by the mg of hydroquinone present. 3) Prepare a calibration curve for gallic acid by plotting the peak area ratio vs. the corresponding weight ratio. A single standard gallic acid solution is used with the intercept set equal to zero to yield a calibration line. Our experience shows that this is valid for peak areas, but may not be valid for peak heights. The tannin calibration is calculated using only the actual tannin content of the standard. Tannin Calibration Curve. 1) From the chromatogram of the mixed tannin standard, sum the areas of the three gallic acids, and any other non-tannin peaks which we define as everything with a retention time less than that of the tri-isomers peak (see Figure 6). 2) Calculate the ratio of the total non-tannin areas summation to the area of the hydroquinone standard peak. 3) Determine the ratio of the calculated weight of the total non-tannin to the weight of internal standard from the gallic acid, calibration curve. 4) Calculate the % total non-tannin:
wt ratio x mg int std X 100 = % non-tannin (2) mg anhydrous tannin sample 5) Determine the % anhydrous tannin in the standard sample by subtracting the % non-tannin from 100. 6) Determine the weight of anhydrous tannin in the standard tannin solution by dividing the % anhydrous tannin by 100 and then multiplying by the weight of anhydrous sample (in mg). 7) Sum the area of the tannin peaks, which we define as everything with a retention time equal to the trigalloyl glucose peak and greater (see Figure 2). 8) Calculate the ratio of the total tannin peaks area summation to the area of the hydroquinone standard peak. 9) Calculate the weight ratio for the total tannin by dividing the weight, in mg, of the anhydrous tannin in the standard solution by the mg of hydroquinone added to the standard solution. 10) Plot the total tannin peaks area ratio vs. the corresponding weight ratio, and draw a straight line through this point and the origin. Assay of Gall Nuts o r Sumac. The ratios of the total non-tannin and total tannin areas to the internal standard 242
Molecular weight
Compound
636.5 788.6 940.7 1092.8 1244.9 1397.0 1549.1 1701.2
1853.3 2005.4
areas are determined as described above under calibration. Read the corresponding weight ratios from the prepared calibration curves and calculate the % anhydrous non-tannin and % anhydrous total tannin, using the following formula: wt % anhydrous (non-tannin or total tannins)
- wt ratio X mg int std X 100
(3) mg sample The above is the manual calculation procedure and is the algorithm used by our Lab Basic program with the HewlettPackard 3352B Lab Data System. Calculation of Weighted Average Molecular Weight of a Tannin Fraction. The individual peak areas of the chromatograms are evaluated by the perpendicular drop technique. For practical consideration, we operated to conserve run time rather than obtain ideal separations. The area of each tannin fraction peak is proportional to the quantity of the component for that particular molecular weight. The proportionate molecular weight fraction is calculated for each component peak by multiplying the corresponding component molecular weight (see Table 111) by the individual peak area and then dividing by the total tannins area. The weighted average molecular weight of the tannins fraction is a summation of the proportionate fraction molecular weights from tri- to dodecagalloyl glucose. CONCLUSION The official assay for tannin is the Hide Powder method (10). This assay determines tannin and non-tannins (which are essentially the gallic acids) by reaction of ground, dried, untanned animal hide with tannin sample solutions. Water is calculated by subtracting the sum of the tannins and nontannins from 100. The results correlate amazingly well with Karl Fischer water determinations. A comparison of the HPLC assay and the Hide Powder procedure was made. The relative standard deviation for the HPLC tannin assay was found to be 2.5% with a range of 4% absolute. The relative standard deviation for the Hide Powder assay was 5.9% with a range of 5.3% absolute. The Hide Powder assay is less precise on purified tannins and can be very inaccurate on samples of gallic acids containing essentially no tannins. The HPLC assay can be completed in about 1h (elapsed time) per sample for purified tannins and ground nut galls or sumac, while the Hide Power assay requires about 2 h actual operator time and about two days elapsed time.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
LITERATURE CITED (1) E. Haslam, "Chemistry of Vegetable Tannins", Academic Press, London, 1966, p 10. (2) E. Haslam, "Chemistry of Vegetable Tannins", Academic Press,London, 1966, pp 106-113.
44, IS33 (1972).
E. Fischer, J. Am. Chem. SOC.,36, 1170 (1914). E. Haslam, R. E. Haworth, S. D. Mills, H. J. Rogers, R. Armitage, and T. Searle, J. Chem. SOC., 1836 (1961). R. Armitage, G.S. Bayliss, J. W. Gramshaw, E.Haslam, R. D. Haworth, K. Jones, H. J. Rogers, and T. Searle, J. Chem. Soc., IS42 (1961). H. W. Zlegler, T. H. Beasley, Sr., and D.W. Smith, J. Assoc. Off. Anal.
( 8 ) W. A. Pearson and G. 0. Spessard. J. Chem. Educ., 52, 814 (1975). (9)R. P. W. Scott and P. Kucera, J. Chromafgr., 83,257 (1973). (10) American Leather Chemlsts Association, Subcommlttee report, J. Am. Leather Chem. Assoc., 51,353(1956).
RECEIVEDfor review July 28,1976. Accepted November 10,
Chem., 58,888 (1975). T. H. Beasley, Sr., H. W. Ziegler, R . L. Charles, and P. King, Anal. Chem.,
1976.
Exclusion Chromatography in Dense Gases: An Approach to Viscosity Optimization J. Calvin Giddings,* Lyle
M. Bowman, Jr.,
and Marcus N. Myers
Department of Chemistry, University of Utah, Salt Lake City, Utah 84 112
Flulds above the normal bolllng point are proposed as solvents for excluslon chromatography (EC) capable of yleldlng reduced vlscoslty and therefore Increased speed. Dense gases constitute the most extreme case of such fluids, and their feaslblllty for EC Is therefore examlned both theoretlcally and experlmentally. The excluslon mechanlsm Is demonstrated for compressed 1,l-dlfluoroethane,and varlous polymers-up to 20 400 MW polystyrene and 40 000 MW polyvlnylpyrolldone-are run In a porous slllca EC column. It Is shown that retentlon is ldentlcal to that found wlth llquld solvents, but that the plate height Is somewhat better. An examlnatlon of VIScoslty data shows that Improvements up to a factor of 5 In vlscoslty rsductlon and chromatographlc operatlng speed are conceivable for columns In whlch adsorptlon Is absent.
Exclusion chromatography (EC), as exemplified in the techniques of gel permeation chromatography and gel filtration chromatography, has had an enormous impact in the characterization of macromolecules (1-3). Consequently, the literature is replete with efforts to improve the resolution, speed, and versatility of the method. In this paper, we hope to add one more dimension to the possible approaches for improving the efficacy of exclusion chromatography. The theoretical basis of the EC technique has been established by the combined work of many authors. The most important conclusion-one that is now quite generally accepted-is that retention is governed by the equilibrium distribution coefficient of solute between bulk solvent and the solvent that occupies the pore space (3-7). Furthermore, this distribution coefficient in ideal EC is determined by entropy factors alone, which reflect the loss of configurational entropy of molecules or particles partitioning into close fitting pores (4, 7 ) .The distribution coefficient is, consequently, unaffected by the nature of the solvent except in those cases in which the solvent has a significant influence on molecular or pore dimensions, or on adsorption. Therefore, as long as the solvent is capable of dissolving the solute, inhibiting its adsorption, and leaving its geometry reasonably intact, the specific nature of the solvent is unimportant insofar as EC retention is concerned. The resolution and speed of EC, in contrast to retention, depend on some of the solvent factors that determine chromatographic efficacy in general (8).The two solvent related parameters that enter these considerations for ideal EC are solvent viscosity and solute-solvent diffusion coefficient. Inasmuch as the latter is inversely proportional to the former for dilute solutions of large solute species with a solvent-in-
dependent geometry, solvent viscosity becomes the single solvent parameter having an important influence on EC optimization under ideal conditions. This matter is discussed in greater detail in an earlier paper dealing with optimization processes in EC (9). In this paper we propose the use of dense gases in place of liquids for the operation of EC columns. Previous work from this laboratory has shown that dense gases-in some cases compressed up to 2000 atm-are capable of taking into solution a variety of macromolecules ranging in molecular weight from lo3 to more than lo5 (10-13), Therefore, there is every reason to expect that dense gases could become perfectly suitable solvents for EC in those cases where solution is possible. We will confirm this expectation shortly. Dense gases used for EC would generally have the advantage of possessing a lower viscosity than equivalent liquids. This viscosity is, of course, a function of the level of compression of the gas. In that solubility is also a function of compression or density, there is a possibility of some degree of trade off between these two parameters for the control of viscosity. By its very nature, dense gas exclusion chromatography must be operated at high pressure. However, moderately high pressures are needed for high efficiency liquid chromatography systems simply to force the fluid through columns of fine particles (8,141. Part of the high pressure of a dense gas EC system can, therefore, be tapped to provide the driving force for flow, thus increasing efficiency. The loss of pressure through the column, however, will be accompanied by some loss in solvent power. Therefore, there will be a trade off between efficiency and solvent power that can, in theory, be adjusted to provide the best separation for a given solute class. In this paper we wish to show, first, that dense gas EC is a realizable concept. Second, we wish to make some comparisons with liquid EC and to analyze the role of viscosity in governing their relative efficiency. In order to accomplish the latter goal, we have developed columns that can be interchanged between dense gas systems and liquid solvent systems. In this way, we are able to isolate solvent effects because the intrinsic column efficiency factors will remain rigorously constant within a given column. The general importance of viscosity as a solvent parameter suggests that this work is only one step in the viscosity related improvement of EC performance. A whole class of methodsincluding the extreme case of dense gases employed here-can be enviaioned as hinging on the use of elevated outlet pressures such that the experimental temperature can exceed the normal boiling point. One approach in this class would involve
ANALYTICAL CHEMISTRY, VOL. 48, NOt 2, FEBRUARY 1977
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